Method and product for regulating cell responsiveness to external signals

ABSTRACT

The present invention relates to isolated MEKK proteins, nucleic acid molecules having sequences that encode such proteins, and antibodies raised against such proteins. The present invention also includes methods to use such proteins to regulate signal transduction in a cell. The present invention also includes therapeutic compositions comprising such proteins or nucleic acid molecules that encode such proteins and their use to treat animals having medical disorders including cancer, inflammation, neurological disorders, autoimmune diseases, allergic reactions, and hormone-related diseases. When MEKK is expressed, it phosphorylates and activates MKKs1-4 (also referred to as MEK-1, MEK-2 and JNKK1 and JNKK2).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/608,890, filed Jun. 30, 2000, which is continuation U.S. Pat. No.6,333,170, issued Dec. 25, 2001, which is a continuation-in-part U.S.Pat. No. 5,753,446, issued on May 19, 1998, which is acontinuation-in-part of U.S. patent application Ser. No. 08/440,421,filed May 12, 1995 (now abandoned), which is a continuation-in-part ofU.S. patent application Ser. No. 08/410,602, filed Mar. 24, 1995 (nowabandoned), which is a continuation-in-part of U.S. Pat. No. 5,854,043,issued on Dec. 29, 1998, which is a continuation-in-part of U.S. Pat.No. 5,405,941, issued on Apr. 11, 1995. The above-referenced patents andpatent applications are incorporated herein by this reference in theirentirety.

GOVERNMENT FUNDING

This invention was made in part with government support under USPHSGrant DK37871 and USPHS Grant GM30324, both awarded by the NationalInstitutes of Health. The government has certain rights to thisinvention.

FIELD OF THE INVENTION

This invention relates to isolated nucleic acid molecules encoding MEKKproteins, substantially pure MEKK proteins, and products and methods forregulating signal transduction in a cell.

BACKGROUND OF THE INVENTION

Mitogen-activated protein kinase (MAPKs) (also called extracellularsignal-regulated kinases or ERKs) are rapidly activated in response toligand binding by both growth factor receptors that are tyrosine kinases(such as the epidermal growth factor (EGF) receptor) and receptors thatare coupled to heterotrimeric guanine nucleotide binding proteins (Gproteins) such as the thrombin receptor. In addition, receptors like theT cell (TCR) and B cell (BCR) receptors are non-covalently associatedwith src family tyrosine kinases which activate MAPK pathways. Speicficcytokines like tumor necrosis factor (TNFα) can also regulate MAPKpathways. The MAPKs appear to integrate multiple intracellular signalstransmitted by various second messengers. MAPKs phosphorylate andregulate the activity of enzymes and transcription factors including theEGF receptor, Rsk 90, phospholipase A₂, c-Myc, c-Jun and Elk-1/TCF.Although the rapid activation of MAPKs by receptors that are tyrosinekinases is dependent on Ras, G protein-mediated activation of MAPKappears to occur through pathways dependent and independent of Ras.

Complementation analysis of the pheromone-induced signaling pathway inyeast has defined a protein kinase system that controls the activity ofSpk1 and Fus3-Kss1, the Schizosaccharomyces pombe and Saccharomycescerevisiae homologs of MAPK (see for example, B. R. Cairns et al., Genesand Dev. 6, 1305 (1992); B. J. Stevenson et al., Genes and Dev. 6, 1293(1992); S. A. Nadin-Davis et al., EMBO J. 7, 985 (1988); Y. Wang et al.,Mol. Cell. Biol. 11, 3554 (1991). In S. cerevisiae, the protein kinaseStep 7 is the upstream regulator of Fus3-Kss1 activity; the proteinkinase Ste11 regulates Step 7. The S. pombe gene products Byr1 and Byr2are homologous to Step 7 and Ste11, respectively. The MEK (MAPK Kinaseor ERK Kinase) or MKK (MAP Kinase kinase) enzymes are similar insequence to Step 7 and Byr1. The MEKs phosphorylate MAPKs on bothtyrosine and threonine residues which results in activation of MAPK. Themammalian serine-threonine protein kinase Raf phosphorylates andactivates MEK, which leads to activation of MAPK. Raf is activated inresponse to growth factor receptor tyrosine kinase activity andtherefore Raf may activate MAPK in response to stimulation ofmembrane-associated tyrosine kinases. Raf is unrelated in sequence toSte11 and Byr2. Thus, Raf may represent a divergence in mammalian cellsfrom the pheromone-responsive protein kinase system defined in yeast.Cell and receptor specific differences in the regulation of MAPKssuggest that other Raf independent regulators of mammalian MEKs exist.

Certain biological functions, such as growth and differentiation, aretightly regulated by signal transduction pathways within cells. Signaltransduction pathways maintain the balanced steady state functioning ofa cell. Disease states can arise when signal transduction in a cellbreaks down, thereby removing the tight control that typically existsover cellular functions. For example, tumors develop when regulation ofcell growth is disrupted enabling a clone of cells to expandindefinitely. Because signal transduction networks regulate a multitudeof cellular functions depending upon the cell type, a wide variety ofdiseases can result from abnormalities in such networks. Devastatingdiseases such as cancer, autoimmune diseases, allergic reactions,inflammation, neurological disorders and hormone-related diseases canresult from abnormal signal transduction.

SUMMARY OF THE INVENTION

The present invention relates to a substantially pure MEKK proteincapable of regulating a MEK kinase dependent pathway. In certainembodiments a MEK kinase comprises a catalytic domain and is capable ofphosphorylating MKK proteins. In preferred embodiments the MEKKsubstrate is selected from the group of MAP kinase kinases consisting ofMEKK1, MKK2, (also called MEK1 and MEK2 respectively) MKK3, or MKK4(also called JNKK1 and JNKK2 or SEK respectively). The present inventionincludes a substantially pure MEKK protein capable of regulating signalsinitiated from a growth factor receptor on the surface of a cell byregulating the activity of MAPK protein. Exemplary MAP kinases includep42, p44, ERK1, ERK2, JNK1, JNK2, or p38 SAPK. In preferred embodimentsa MEK kinase can activate at least one of the group c myc, cJun, cPLA2,Rsk 90, TCF, Elk-1, or ATF-2.

In certain embodiments the MEKK protein of the present invention isregulates the activity of a MAPK protein independently of Raf. Inpreferred embodiments the MEKK proteins described herein are capable ofbinding members of the Ras superfamily. Exemplary polypeptides whichbind to MEKK proteins include Ras, Rac/Cdc42, or Rho.

In particular, the substantially pure MEKK proteins of the presentinvention comprise at least a portion of an amino acid sequence shown inone of SEQ ID Nos:2, 4, 6, 8, 10, 12, or 14. In other embodiments,proteins at least 50% homologous, at least 75% homologous, preferably atleast 85% homologous, or more preferably 95% homologous to one of SEQ IDNos: 2, 4, 6, 8, 10, 12, or 14 are also contemplated.

In certain embodiments MEKK proteins have homology to the kinasecatalytic domain of one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, or 14. Inother embodiments proteins having at least 50% homology, at least 75%homology, preferably at least 85% homology, or more preferably at least95% homology to the kinase catalytic domain of one of SEQ ID Nos: 2, 4,6, 8, 10, 12, or 14 are contemplated. In more preferred embodiments thekinase domain of a MEKK protein is capable of phosphorylating a MAPkinase kinase protein and binding to a member of the ras superfamily,such as ras or rac or cdc42 protein.

In another embodiment the MEKK protein of the present inventioncomprises a NH2 regulatory domain represented in one of SEQ ID Nos: 2,4, 6, 8, 10, 12, or 14. In other embodiments MEKK proteins whichcomprise regions of at least 50% homology, at least 75% homology,preferrably 85% homology, or more preferably at least 95% homology tothe NH2 regulatory domain of one of SEQ ID Nos 2, 4, 6, 8, 10, 12, or 14are contemplated.

In a further embodiment MEKK proteins which have molecular weightsranging from 60 to 190 are contemplated. Preferred molecular weights are98 kD for MEKK1, 69.5 kD for MEKK2, 71 kD for MEKK3, and 95-98 kD forMEKK 4. In other embodiments MEKK 4 migrates with an apparent molecularweight of 185 kD.

MEKK proteins of the present invention lack an SH2 or SH3 domain. Inpreferred embodiments exemplary MEKK proteins comprise a proline richSH3 binding motif. In certain embodiments, MEKK proteins of the instantinvention comprise a Pleckstrin homology domain.

In a particularly preferred embodiment, exemplary MEKK proteins cancompetitively inhibit the activity of a MEKK designated in one or moreof SEQ ID Nos: 2, 4, 6, 8, 10, or 12, or 14.

Fragments of MEKK proteins are also contemplated by the presentinvention. In preferred embodiments exemplary MEKK proteins lack a MEKKregulatory domain. In particularly preferred embodiments MEKK proteinfragments lack the serine/threonine rich regulatory domain shown in oneof SEQ ID Nos: 2, 4, 6, 8, 10, 12, or 14. In another embodiment thefragment of a MEKK protein lacks the serine/threonine kinase domain of aMEKK protein. In preferred embodiments MEKK protein fragments lack theserine/threonine kinase domain shown in one of SEQ ID Nos: 2, 4, 6, 8,10, 12, or 14.

In another embodiment the MEKK protein of the present invention is afusion protein further comprising, in addition to the MEKK polypeptide,a second polypeptide sequence having an amino acid sequence unrelated toMEKK polypeptide sequence. In a preferred embodiment the fusion proteinincludes as a second polypeptide sequence, a polypeptide which functionsas a detectable label for detecting the presence of said fusion proteinor as a matrix-binding domain for immobilizing said fusion protein.

In another embodiment a MEKK protein or a portion of a MEKK proteinwhich is encoded by a nucleic acid sequence that is capable ofhybridizing under stringent conditions with a nucleic acid moleculeencoding an amino acid sequence including SEQ ID Nos: 2, 4, 6, 8, 10,12, or 14. The substantially pure MEKK protein capable of regulating theactivity of a MEKK dependent pathway, said protein having an amino acidsequence distinct from Raf protein.

In a particularly preferred embodiment the MEKK protein of the presentinvention is capable of regulating apoptosis in a cell.

One aspect of the present invention includes an isolated nucleic acidmolecule having a sequence encoding a protein capable of regulating aMEKK dependent pathway. In preferred embodiments the nucleic acid of thepresent invention encodes a protein which phosphorylates a MAP kinasekinase independently of Raf protein and is capable of regulating theactivity of MAPK protein. In particular, the present invention includesan isolated nucleic acid molecule shown in one of SEQ ID Nos: 1, 3, 5,7, 9, 11, or 13. In another embodiment nucleic acids capable ofhybridizing under stringent conditions with a nucleic acid sequenceselected from the group consisting of SEQ ID Nos: 1, 3, 5, 7, 9, 11, or13 are also contemplated.

In certain embodiments the nucleic acid of the present invention encodesa protein which regulates a MAP kinase kinase selected from the groupconsisting of p42, p44, ERK1, ERK2, JNK1, JNK2, or p38 SAPK.

In another embodiment nucleic acids at least 50%, at least 75%, morepreferably at least 85%, or most preferably 95% homologous to one of SEQID Nos: 1, 3, 5, 7, 9, 11, or 13 are also contemplated.

In another embodiment the nucleic acid of the present invention encodesa polypeptide, wherein said polypeptide i) phosphorylates a MAP kinasekinase protein and ii) binds to a ras superfamily protein. In certainembodiments the ras superfamily member is ras and said binding ismediated by the carboxy terminus of said polypeptide. In anotherembodiment the nucleic acid encodes a protein with a cdc42/rac bindingsite.

In another embodiment the nucleic acid of the present invention encodesa polypeptide which comprises a MKK consensus binding site. In anotherembodiment the nucleic acid of the present invention encodes apolypeptide which comprises a proline rich SH3 binding motif.

In another embodiment the nucleic acid of the present invention iscapable of hybridizing under stringent conditions to a nucleic acidprobe having a sequence represented by at least 60 consecutivenucleotides of sense of antisense of one or more of SEQ ID Nos:1, 3, 5,7, 9, 11, or 13. Oligonucleotide probes which hybridize to one of SEQ IDNos:1, 3, 5, 7, 9, 11, or 13 are also contemplated.

Another aspect of the present invention includes a recombinant molecule,comprising a nucleic acid molecule capable of hybridizing understringent conditions with a nucleic acid sequence including SEQ ID Nos:1, 3, 5, 7, 9, 11, or 13 in which the nucleic acid molecule isoperatively linked to an expression vector.

In another embodiment a nucleic acid of the present invention ispreferably linked to a transcriptional regulatory sequence and said geneconstruct is deliverable to a cell and causes the cell to be transfectedwith said gene construct.

Yet another aspect of the present invention is a recombinant celltransformed with a recombinant molecule, comprising a nucleic acidmolecule operatively linked to an expression vector, the nucleic acidmolecule comprising a nucleic acid sequence capable of hybridizing understringent conditions with a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13.

In another embodiment the present invention comprises a host celltransfected with the expression vector comprising one of SEQ ID Nos: 1,3, 5, 7, 9, 11, or 13. Another embodiment of the present inventioncomprises a method for producing recombinant MEKK polypeptide byculturing a host cell transfected with such an expression vector.

Also contemplated by the present invention are transgenic animals havingcells which harbor a transgene encoding a MEKK polypeptide or in which agene for a MEKK is disrupted.

One embodiment of the invention provides for drug screening assays thatcan be used to identify compounds which inhibit the interaction of MEKKwith a MEKK binding protein, said binding protein including a substrateor upstream activator of MEKK as described herein. The invention furthercontemplates the development of peptides or mimetics or nucleic acidswhich can block MEKK activation in a similar manner. In a preferredembodiment a peptide which blocks the interaction of a MEKK protein withRac or Cdc42 is provided. In a further preferred embodiment a peptidewhich blocks the interaction of a MEKK protein with Ras is alsoprovided.

The present invention also includes a method for regulating thehomeostasis of a cell comprising regulating the activity of aMEKK-dependent pathway relative to the activity of a Raf-dependentpathway in the cell. In particular, the method comprises regulating theapoptosis of the cell. Such a method is useful for the treatment of amedical disorder. In particular, the method is useful for inhibitingtumorigenesis and autoimmunity.

According to the present invention, the method for treatment of adisease, comprises administering to a patient an effective amount of atherapeutic compound comprising at least one regulatory moleculeincluding a molecule capable of decreasing the activity of aRaf-dependent pathway, a molecule capable of increasing the activity ofa MEKK-dependent pathway, and combinations thereof, in which theeffective amount comprises an amount which results in the depletion ofharmful cells involved in the disease.

Also included in the present invention is a therapeutic compound capableof regulating the activity of a MEKK-dependent pathway in a cellidentified by a process, comprising: (a) contacting a cell with aputative regulatory molecule; and (b) determining the ability of theputative regulatory compound to regulate the activity of aMEKK-dependent pathway in the cell by measuring the activation of atleast one member of said MEKK-dependent pathway.

One embodiment of the present invention includes a substantially pureprotein, in which the protein is isolated using an antibody capable ofselectively binding to a MEKK protein capable of phosphorylatingmammalian MKK proteins and capable of regulating the activity of MAPKproteins independent of Raf protein, the antibody capable of beingproduced by a method comprising: (a) administering to an animal aneffective amount of a substantially pure MEKK protein of the presentinvention; and (b) recovering an antibody capable of selectively bindingto the MEKK protein.

Another embodiment of the present invention includes an isolatedantibody capable of selectively binding to a MEKK protein, the antibodycapable of being produced by a method comprising administering to ananimal an effective amount of a substantially pure protein of thepresent invention, and recovering an antibody capable of selectivelybinding to the MEKK protein. Also contemplated by the present inventionis a MEKK polypeptide bound by an antibody which specifically binds to aMEKK protein shown in one of SEQ ID Nos: 2, 4, 6, 8, 10, or 12.

This invention further relates to biological responses modulated by theMAPK pathway, which is regulated by signaling through interactions ofRas protein and MEK kinase (MEKK) protein. These biological responsesinclude activation of immune responses, especially in B cells and in Tcells; other biological responses regulated by the Ras protein; MEKkinase (MEKK) interactions including activation, proliferation andimmunoglobulin class switching. Methods herein disclosed may be used tospecifically modulate the interaction of Ras protein and MEK kinase(MEKK) protein, or to identify compounds which specifically act to alterthe interaction of Ras protein and MEK kinase protein. Alternatively,such biological responses regulated by the interaction of Ras proteinand MEK kinase (MEKK) protein may be manipulated to achieve therapeuticresults in vivo by methods of the present invention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the signal pathways ofvertebrates and yeast.

FIG. 2 is a schematic representation of the dual MEKK and Raf pathwaysdivergent from Ras protein pathway.

FIG. 3 shows the activation of MAPK in COS cells transfected with MEKK.

FIG. 4 shows the activation and phosphorylation of MEK in COS cellstransfected with MEKK.

FIG. 5 shows the relative ability of immunoprecipitated MEKK and Raf-Bto phosphorylate kinase inactive MEK-1.

FIG. 6 shows a time course of EGF-stimulated MEKK and Raf-B activation.

FIG. 7 shows that the immunodepletion of Raf-B from MEKKimmunoprecipitates has no effect on MEKK activity.

FIG. 8 shows that the immunodepletion of Raf-B from MEKKimmunoprecipitates decreases Raf-B activity.

FIG. 9 shows inhibition of MEKK and Raf-B activation by dominantnegative N¹⁷RAS expression.

FIG. 10 shows inhibition of EGF activation of MEKK by forskolin.

FIG. 11 shows improved MEKK activity by truncated MEKK molecules.

FIG. 12 shows JNK activation by MEKK protein.

FIG. 13 shows regulation of c-Myc controlled transcription and not CREBcontrolled transcription by MEKK protein.

FIG. 14 is a schematic representation of MEKK regulation of c-Myccontrolled transcription.

FIG. 15 shows wild type Swiss 3T3 cells transfected with pCMV5BXBRaf orpCMV5 without a cDNA insert in the presence of expression plasmidsencoding GA14/Elk-1 and Gal4-TK-luciferase. Cells were lysed and assayedfor luciferase activity 48 hours post-transfection.

FIG. 16. Induction of MEKK_(COOH) expression by IPTG in Swiss 3T3 cellsincreases the number of condensed cells and stimulates c-Myctransactivation. In panel A, cells were incubated in the presence orabsence of 5 mM IPTG for forty eight hours. Cells were stained withacrodine orange and condensed cells quantitated per 1000 cells countedper coverslip. In panel B Swiss 3T3 cells with inducible MEKK_(COOH)were incubated in the presence or absence of IPTG. The indicated cellswere then exposed to UV-C irradiation and then fixed and stained withpropidium iodide. The percentage of apoptotic cells was enumerated.

FIG. 17 shows that MEKK_(COOH) stimulates JNK/SAPKm but did not activateERK (p42/44 MAPK) or p38Hog1. Induction of MEKK_(COOH) does not activateERK or p38, whereas PDGF or sorbitol, (used as positive controls) do.Activation of the cells with PDGF or sorbitol activated ERK and p38/Hog1as a control.

FIG. 18 shows that induction of MEKK_(COOH) expression did notsignificantly increase Gal4/Jun transactivation (left panel). Transienttransfection of MEKK_(COOH) resulted in increased Gal4/Juntransactivation in the MEKK2 Swiss 3T3 clone (right panel).

FIG. 19 shows that competitive inhibitory JNK/SAPK(APF) attenuatesGA14/Jun but not Gal4/myc activation. The results are representative ofthree independent experiments where a three-fold excess of JNK/SAPK(APF)inhibited approximately 65% of Gal4/Jun activation with no effect onGal4/myc activation.

FIG. 20 shows the induction of apoptosis in L929 cells expressingMEKK_(COOH) domain by TNF.

FIG. 21 shows similar stimulation of MAPK activity by MEKK protein andRaf protein.

FIG. 22 is a graph illustrating the ability of various MEKK proteins,and fragments thereof, to activate a JNK activity.

FIG. 23 is a graph illustrating the ability of various MEKK proteins,and fragments thereof, to activate ERK1 and ERK2.

FIG. 24 This figure shows that TNF induces apoptosis in L929 cells andthat this effect is blocked by bFGF. In panel A cells were treated withthe indicated concentrations of TNFα for 15 hours and were assayed foruptake of neutral red. In panel B cells were untreated (solid bars),treated with 0.5 ng/ml bFGF (dotted bars) or 5.0 ng/ml bFGF (hatchedbars) and the indicated concentrations of TNFα for 18 hours. Cellviability was assessed by neutral red assay.

FIG. 25 shows the activation of JNK and MAPK in L929 cells. In panel Acells were treated for 10 minutes with the indicated concentration ofTNFα. JNK activation was measured using a solid phase kinase assayresulting in phosphorylation of GST-Jun. In panel C the time course ofMAPK activation is shown. MAPK was isolated from cell lysates on DEAEsephacel columns and MAPK activation was measured by phosphorylation ofthe EGFR peptide substrate. Panel C depicts the concentration curve ofMAPK activation by TNFα. Cells were treated with the indicatedconcentration of TNFα and MAPK was assayed.

FIG. 26 depicts the activation of MAPK by bFGF in L929 cells. Serumstarved L929 cells were stimulated for 10 min with the indicatedconcentration of bFGF.

FIG. 27 shows that bFGF does not inhibit TNFα stimulation of JNKactivity. In panel A serum starved L929 cells were treated as indicated.Radiolabel incorporated into GST-Jun is expressed in arbitraryphosphorimaging units. In panel B cells were stimulated as indicated andassayed for MAPK activity.

FIG. 28 shows the effect of dominant negative N17 Ras or constitutivelyactive V12 Ras on MAPK and JNK activities. In panel A cells wereuninduced (−) or induced (+) to express N17 Ras by overnight treatmentwith 5 mM IPTG. The cells were unstimulated (−) or stimulated (+) for 10min with 0.5 ng/ml bFGF. MAPK activity was assayed. In panel B 41.LAC1or V12 Ras cells were induced with IPTG, stimulated as indicated andanalyzed for MAPK activation.

FIG. 29 shows the effect of N17 Ras on TNFα killing and bFGF protection.Ras expression was induced with 5 mM IPTG for 10 hours and cells weresubsequently treated with 5 ng/ml TNFα in the presence or absence of 0.5ng/ml bFGF for 16 hours. Cells were fixed and stained with propidiumiodide. The percentage of apoptotic cells was calculated. Solid barsrepresent cells induced with IPTG; hatched bars, induced with IPTG andtreated with TNFα; checked bars, induced with IPTG and treated with TNFαand bFGF.

FIG. 30 shows the inhibition of MAPK activity and elimination of thebFGF protective effect of treatment with the MEK-1 inhibitor PD #098059.In panel A serum starved L929 cells were untreated or treated for 1 hourat 37° C. with the MEK-1 inhibitor (PD) and then unstimulated orstimulated with bFGF. MAPK activity was measured. In panel B L929 cellswere untreated or treated for 1 hour at 37° C. with PD and then wereuntreated or treated with TNFα alone or in combination with bFGF for 18hours. Cell viability was assessed by neutral red assay.

DETAILED DESCRIPTION OF THE INVENTION

Through a series of inducible and reversible protein-proteininteractions and phosphorylation-mediated enzymatic activities,regulatory proteins are recruited to relay signals throughout the cell.Such interactions are involved in all stages of the intracellular signaltransduction process—at the plasma membrane, where the signal isinitiated; in the cytoplasm, where the signals are disseminated todifferent cellular locations; and in the nucleus, where other proteinsinvolved in transcriptional control form complexes to regulatetranscription of particular genes. The structural nature of proteininteractions and control of enzymatic activities in signal transductionis emerging through the identification of the individual proteins thatparticipate in each signal transduction pathway, the elucidation of thetemporal order in which these proteins interact, and the definition ofthe sites of contact between the proteins. The understanding gained inintracellular signaling pathways of cells will be advantageous indeveloping the next generation of pharmaceuticals. In particular, thepleiotropic richness of intracellular signaling pathways in cellsrepresents a means for developing more selective pharmacologicalactivity in a therapeutic agent than may be possible in the presentgeneration of drugs.

The present invention concerns the discovery of a family of novelmitogen ERK kinase kinase proteins (referred to herein as “MEK kinases”,“MEKKs” or “MEKK proteins”) which function in intracellular signaltransduction pathways in a variety of cells, and accordingly have a rolein determining cell/tissue fate and maintenance. The family of MEKKgenes or gene products provided by the present invention apparentlyconsists of at least six different members (MEKK 4.2 is a splicingvariant of MEKK4.1 and MEKK 2.2 is a sequencing variant of MEKK2) withample evidence indicating that yet other members of the family exist.

A salient feature of the MEKK gene products deriving from this discoverynot only implicates these proteins in intracellular signaling, but alsostrongly suggests that the diversity of the MEKK family is important toproviding a diversity of responses to different environmental cues. Thatis, the ability of a cell to respond to a particular growth factor,morphogen, or even stress cue, and the type of response the cellundergoes is dependent at least in part upon which MEKK gene productsare expressed in the cell and/or engaged by signals propagated upstreamof the kinase.

Still another important feature of the present invention is thediscovery of the involvement of MEKK proteins in certain apoptoticpathways.

Accordingly, certain aspects of the present invention relate to nucleicacids encoding vertebrate MEKK proteins, the MEKK proteins themselves,antibodies immunoreactive with MEKK proteins, and preparations of suchcompositions. Moreover, the present invention provides diagnostic andtherapeutic assays and reagents for detecting and treating disordersinvolving, for example, aberrant expression or activation of the MEKKgene products. In addition, drug discovery assays are provided foridentifying agents which can modulate the biological function of MEKKproteins, such as by altering the binding of the protein to eitherdownstream or upstream elements in a signal transduction pathway, orwhich inhibit the kinase activity of the MEKK protein. Such agents canbe useful therapeutically to alter the growth and/or differentiation ofa cell. Other aspects of the invention are described below or will beapparent to those skilled in the art in light of the present disclosure.

Initial cloning of a member of the mammalian MEKK family wasaccomplished using primers based on sequences for the yeast proteinkinases Byr2 (from S. pombe) and Ste11 (from S. cerevisiae). Using thesequence obtained for the mammalian MEKK cDNA, other MEKK transcriptshave been detected and several subsequently cloned to reveal a family ofmammalian MEKK proteins.

TABLE 1 Guide to MEKK sequences in Sequence Listing Nucleotide AminoAcid MEKK1.1 SEQ ID No. 1 SEQ ID No. 2 MEKK1.2 SEQ ID No. 3 SEQ ID No. 4MEKK2.1 SEQ ID No. 5 SEQ ID No. 6 MEKK2.2 SEQ ID No. 7 SEQ ID No. 8MEKK3 SEQ ID No. 9 SEQ ID No. 10 MEKK4.1 SEQ ID No. 11 SEQ ID No. 12MEKK4.2 SEQ ID No. 13 SEQ ID No. 14

The foregoing SEQ ID NO's represent sequences deduced according tomethods disclosed in the Examples. It should be noted that since nucleicacid and amino acid sequencing technology is not entirely error-free,the foregoing SEQ ID NO's, at best, represent apparent nucleic acid andamino acid sequences of MEKK proteins of the present invention. Forconvenience, we will use the term MEKK1 to refer to both MEKK1.1 andMEKK 1.2, MEKK 2 to refer to both MEKK2.1 and MEKK 2.2, and MEKK4 torefer to both MEKK4.1 and MEKK 4.2 herein.

The primary sequence of the MEKK proteins suggests two functionaldomains, an amino-terminal moiety rich in serine and threonine thatapparently serves a regulatory role, and a carboxy-terminal proteinkinase catalytic domain. The catalytic domain of, for example, MEKK1shows approximately 35 percent identity with the amino acid sequences ofthe catalytic domains of Byr2 and Ste11. The amino-terminal moieties ofeach of the mammalian MEKKs show little similarity with Ste11 or Byr2.

Furthermore, the MEKK family is apparently encoded by several genes, atleast some of which are able to produce different transcripts bydifferential splicing. For example, the divergence in sequence amongstthe catalytic domains of each of MEKK1 to MEKK4 indicated that separategenomic genes encode each paralog. However, MEKK2 and MEKK4 genes cangive rise to at least two different transcripts, presumably bedifferential splicing. Expression data suggests that MEKKs 1-4 areubiquitously expressed.

By use of overexpression and/or constitutively activated MEKKs, avariety of cellular substrates for each MEKK protein have beenidentified. In general, the proteins of the MAP kinase kinases (MEK)family are each targets for one or more of the MEKKs. Moreover, the dataset out below demonstrate that MEKK-dependent signal propagation canresult in the phosphorylation/activation of members of the MAP kinasefamily, such as p42MAPK, p44MAPK, p38MAPK, and the Jun NH₂-terminalkinases (JNKs).

Certain of the MEKK proteins have been shown to be activated, e.g., askinases, in response to growth factors and cytokines (such as TNFα andchemoattractants like FMLP and IL-8) and other environmental cues,including stress, as well as expression of activated Ras or othermembers of the Ras Superfamily, including Rac and Cdc42. It isdemonstrated below that the kinase domain of at least MEKK1 binds toactivated Ras in a GTP-dependent manner, implicating that interaction asa potential therapeutic target. Moreover, a Ras effector domain peptideblocks the binding of the MEKK catalytic domain with the GTP-bound formof Ras. In addition, it is shown in the appended Examples that MEKK4binds to Rac, a low molecular weight GTP binding protein of the Rassuperfamily. The sequence of MEKK4 which binds to Cdc42 and Rac has beenidentified. This sequence IIGQVCDTPKSYDNVMHVGLR occurs around residue1306-1326 of MEKK4.2 or 599-619 of MEKK4 and peptides from this regioncan be used to block the binding of the MEKK catalytic domain with Cdc42and Rac.

Yet another set of experimental data provided in the appended examplesindicates that activation of certain MEKK pathways can lead toapoptosis. The integration of signal transduction pathways regulated bygrowth factor and cytokine receptors commits a cell either toproliferation or apoptosis (Sumimoto, S. L. et al. (1994) J. Immunol.153:2488-2496). Specific cytokines and stresses to cells, such as DNAdamage, appear to preferentially activate the JNK/SAPK pathway whichleads to apoptosis. Several checkpoints exist in the pathways leading toapoptosis that involve proteins such as Bcl2 and p58, which can bothinhibit apoptosis. The MEKK proteins are therefore, important to thedynamic balance between growth factor-activated ERK and stress-activatedJNK/p38 pathways and accordingly important in determining whether a cellsurvives or undergoes apoptosis. To date candidate molecules involved insignaling apoptosis include ceramide, Ras, Rho, c-myc, c-Jun, and theproteins associated with the TNFα receptor and Fas.

One aspect of the present invention relates to isolated MEKK proteins.As used herein protein, peptide, and polypeptide are meant to besynonomous. According to the present invention, an isolated protein is aprotein that has been removed from its natural milieu. It will beunderstood that “isolated”, with respect to MEKK polypeptides, is meantto include formulations of the polypeptides which are isolated from, orotherwise substantially free of other cellular proteins (“contaminatingproteins”), especially other cellular signal transduction factors,normally associated with the MEKK polypeptide. Thus, isolated MEKKprotein preparations include preparations having less than 20% (by dryweight) contaminating protein, and preferably having less than 5%contaminating protein (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 5000, can bepresent). Functional forms of the subject MEKK polypeptides can beprepared, for the first time, as purified preparations by using a clonedgene as described herein. Alternatively, the subject MEKK polypeptidescan be isolated by affinity purification using, for example, acatalytically inactive MEK. “Isolated” does not encompass either naturalmaterials in their native state or natural materials that have beenseparated into components (e.g., in an acrylamide gel) but not obtainedeither as pure (e.g. lacking contaminating proteins, or chromatographyreagents such as denaturing agents and polymers, e.g. acrylamide oragarose) substances or solutions.

An isolated MEKK protein can, for example, be obtained from its naturalsource, be produced using recombinant DNA technology, or be synthesizedchemically. As used herein, an isolated MEKK protein can be afull-length MEKK protein or any homologue of such a protein, such as aMEKK protein in which amino acids have been deleted (e.g., a truncatedversion of the protein, such as a peptide), inserted, inverted,substituted and/or derivatized (e.g., by glycosylation, phosphorylation;acetylation, myristoylation, prenylation, palmitoylation, amidationand/or addition of glycosylphosphatidyl inositol), wherein the modifiedprotein is capable of phosphorylating MAP kinase kinases, such asmitogen ERK kinases (MEKs (MKK1 and MKK2)) and/or Jun kinase kinases(JNKKs (MKK3 and MKK4)).

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding one of the MEKKpolypeptides of the present invention, including both exon and(optionally) intron sequences. A “recombinant gene” refers to nucleicacid encoding a vertebrate MEKK polypeptide and comprising vertebrateMEKK-encoding exon sequences, though it may optionally include intronsequences which are either derived from a chromosomal vertebrate MEKKgene or from an unrelated chromosomal gene. Exemplary recombinant genesencoding the subject vertebrate MEKK polypeptide are represented in theappended Sequence Listing. The term “intron” refers to a DNA sequencepresent in a given vertebrate MEKK gene which is not translated intoprotein and is generally found between exons.

A homologue of a MEKK protein is a protein having an amino acid sequencethat is sufficiently similar to a natural MEKK protein amino acidsequence that a nucleic acid sequence encoding the homologue is capableof hybridizing under stringent conditions to (i.e., with) a nucleic acidsequence encoding the natural MEKK protein amino acid sequence. As usedherein, stringent hybridization conditions refer to standardhybridization conditions under which nucleic acid molecules, includingoligonucleotides, are used to identify similar nucleic acid molecules.Such standard conditions are disclosed, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press,1989. A homologue of a MEKK protein also includes a protein having anamino acid sequence that is sufficiently cross-reactive such that thehomologue has the ability to elicit an immune response against at leastone epitope of a naturally-occurring MEKK protein.

The minimal size of a protein homologue of the present invention is asize sufficient to be encoded by a nucleic acid molecule capable offorming a stable hybrid with the complementary sequence of a nucleicacid molecule encoding the corresponding natural protein. As such, thesize of the nucleic acid Molecule encoding such a protein homologue isdependent on nucleic acid composition, percent homology between thenucleic acid molecule and complementary sequence, as well as uponhybridization conditions per se (e.g., temperature, salt concentration,and formamide concentration). The minimal size of such nucleic acidmolecules is typically at least about 12 to about 15 nucleotides inlength if the nucleic acid molecules are GC-rich and at least about 15to about 17 bases in length if they are AT-rich. As such, the minimalsize of a nucleic acid molecule used to encode a MEKK protein homologueof the present invention is from about 12 to about 18 nucleotides inlength. There is no limit, other than a practical limit, on the maximalsize of such a nucleic acid molecule in that the nucleic acid moleculecan include a portion of a gene, an entire gene, or multiple genes, orportions thereof. Similarly, the minimal size of a MEKK proteinhomologue of the present invention is from about 4 to about 6 aminoacids in length, with preferred sizes depending on whether afull-length, multivalent protein (i.e., fusion protein having more thanone domain each of which has a function), or a functional portion ofsuch a protein is desired.

MEKK protein homologues can be the result of allelic variation of anatural gene encoding a MEKK protein. A natural gene refers to the formof the gene found most often in nature. MEKK protein homologues can beproduced using techniques known in the art including, but not limitedto, direct modifications to a gene encoding a protein using, forexample, classic or recombinant DNA techniques to effect random ortargeted mutagenesis. As will be understood, mutagenesis includes pointmutations, as well as deletions and truncations of the MEKK polypeptidesequence. The ability of a MEKK protein homologue to phosphorylate MEKand/or JNKK protein can be tested using techniques known to thoseskilled in the art. Such techniques include phosphorylation assaysdescribed in detail in the Examples section.

With respect to homologues, it will also be possible to modify thestructure of the subject MEKK polypeptides for such purposes asenhancing therapeutic or prophylactic efficacy, or stability (e.g., exvivo shelf life and resistance to proteolytic degradation in vivo). Suchmodified polypeptides, when designed to retain at least one activity ofthe naturally-occurring form of the protein, are considered functionalequivalents of the MEKK polypeptide described in more detail herein.Such modified peptide can be produced, for instance, by amino acidsubstitution, deletion, or addition.

In one embodiment, a MEKK protein of the present invention is capable ofregulating a MEKK-dependent pathway. According to the present invention,a MEKK-dependent pathway refers generally to a pathway in which a MEKKprotein regulates a pathway substantially independent of Raf, though thepathway including the MEKK protein may converge with common members of apathway involving Raf protein, such as a MEK protein (see FIG. 1).

In certain preferred embodiments, the MEKK protein will be involved in apathway controlling the phosphorylation of a mitogen-activated protein(MAP) kinase. The mammalian MAP kinase family includes, for example, theextracellular signal-regulated protein kinases (ERK1 and ERK2), p42 orp44 MAPKs. In another preferred embodiment the MEKK protein will beinvolved in the pathway controlling c-Jun NH2-terminal kinases (JNKs, orSAPKs), and the so-called “p38 subgroup” kinases (p38 and Hog-1kinases). For example, it is contemplated that the MEKK proteins of thepresent invention interact with, and directly phosphorylate members ofthe MAP kinase kinase family (MEKs or MKKs), as MEK1, MEK2, MKK1, MKK2,or the stress-activated kinases (SEKs), and the Jun kinase kinases(JNKK1, JNKK2, MKK3, MKK4), or the like.

An exemplary MEKK-dependent pathway includes a pathway involving a MEKKprotein and a MKK protein. One of skill in the art can determine whetheror not the regulation of a pathway by a MEKK protein is substantiallyindependent of a Raf protein by comparing the ability of a MEKK proteinand a Raf protein to regulate the phosphorylation of a downstream memberof such pathway using, for example, the general method described inExample 16. For instance, a MEKK protein can regulate a pathwaysubstantially independently of a Raf protein if the MEKK protein inducesphosphorylation of a member of the pathway downstream of MEKK (e.g.,proteins including JEK, Jun kinase, Jun and/or ATF-2) by an amountsignificantly greater than that seen when Raf protein is utilized. Raf-1and B-Raf kinases selectively regulate MEK1 and MEK2 and do notrecognize the JNKK proteins, thus Raf proteins appear to be highlyselective for the regulation of p42/p44 MAPK pathways. MEKK proteins, incontrast, are capable of regulating both JNK and p42/p44 MAPK pathways.

For example, MEKK induction of phosphorylation of a JNK protein ispreferably at least about 10-fold, more preferably at least about20-fold and even more preferably at least about 30-fold than thephosphorylation of the JNK protein induced when using a Raf protein. IfMEKK induction of phosphorylation is similar to Raf protein induction ofphosphorylation, then one of skill in the art can conclude thatregulation of a pathway by a MEKK protein includes members of a signaltransduction pathway that could also include Raf protein. For example,MEKK induction of phosphorylation of MAPK is of a similar magnitude asinduction of phosphorylation with Raf protein.

A “Raf-dependent pathway” refers to a signal transduction pathway inwhich a Raf protein regulates a signal transduction pathwaysubstantially independently of a MEKK protein, and a pathway in whichRaf protein regulation converges with common members of a pathwayinvolving MEKK protein. The independence of regulation of a pathway by aRaf protein from regulation of a pathway by a MEKK protein can bedetermined using methods similar to those used to determine MEKKindependence.

In another embodiment, a MEKK protein is capable of regulating theactivity of signal transduction proteins including, but not limited to,mitogen activated ERK kinases (MEKs), mitogen activated protein kinases(MAPKs), transcription control factor (TCF), Ets-like-1 transcriptionfactor (Elk-1), Jun ERK kinases (JNKKs), Jun kinases (JNK; which isequivalent to SAPK), stress activated MAPK proteins, Jun, activatingtranscription factor-2 (ATF-2) and/or Myc protein. As used herein, the“activity” of a protein can be directly correlated with thephosphorylation state of the protein and/or the ability of the proteinto perform a particular function (e.g., phosphorylate another protein orregulate transcription). Preferred MEK proteins regulated by a MEKKprotein of the present invention include MEK-1 and/or MEK-2 (MKK1 orMKK2). Preferred MAPK proteins regulated by a MEKK protein of thepresent invention include p38/Hog-1 MAPK, p42 MAPK and/or p44 MAPK.Preferred stress activated MAPK proteins regulated by a MEKK protein ofthe present invention include Jun kinase (JNK), stress activated MAPK-αand/or stress activated MAPK-β. A preferred MEKK protein that is capableof activating p42/44 MAPK proteins includes a protein encoded by thenucleic acid sequence represented by SEQ ID NO:9 with a protein havingthe amino acid sequence represented by SEQ ID NO:10 being morepreferred. A preferred MEKK protein that is capable of activating JNKMAPK is encoded by the nucleic acid sequence represented by one of SEQID Nos: 5 or 7, with a protein having the amino acid sequencerepresented by one of SEQ ID Nos: 6 or 8 being more preferred.

A MEKK protein of the present invention is capable of increasing theactivity of an MEK protein over basal levels of MEK (i.e., levels foundin nature when not stimulated). For example, a MEKK protein ispreferably capable of increasing the phosphorylation of an MEK protein(such as MEK1 or MEK2, also known as MKK1 and MKK2 respectively) by atleast about 2-fold, more preferably at least about 3-fold, and even morepreferably at least about 4-fold over basal levels when measured underconditions described in Example 9. In another embodiment, a preferredMEKK protein is capable of increasing the phosphorylation of a JNKKprotein (such as JNKK1 or JNKK2, also known as MKK3 and MKK4respectively).

A preferred MEKK protein of the present invention is also capable ofincreasing the activity of an MAPK protein over basal levels of MAPK(i.e., levels found in nature when not stimulated). For example, a MEKKprotein of the present invention is preferably capable of increasingMAPK activity at least about 2-fold, more preferably at least about3-fold, and even more preferably at least about 4-fold over basalactivity when measured under the conditions described in Example 3.

Moreover, a MEKK protein of the present invention is capable ofincreasing the activity of a JNK protein. JNK regulates the activity ofthe transcription factor JUN which is involved in controlling the growthand differentiation of different cell types, such as T cells, neuralcells or fibroblasts. JNK also regulates Elk-1, an Ets family member.JNK shows structural and regulatory homologies with MAPK. For example, aMEKK protein of the present invention is preferably capable of inducingthe phosphorylation of JNK protein at least about 30 times more thanRaf, more preferably at least about 40 times more than Raf, and evenmore preferably at least about 50 times more than Raf, when measuredunder conditions described in Example 16.

In addition, a MEKK protein of the present invention is capable ofspecific binding to a Ras superfamily protein. In particular, a MEKKprotein is capable of binding to a Ras protein that is associated withGTP. According to the present invention, a MEKK protein binds to Ras viathe COOH terminal region of the MEKK protein, e.g., a ras-bindingdomain. A preferred MEKK protein that is capable of binding to Ras or amember of the ras superfamily is endoced by the nucleic acid shown inSEQ ID No:1, 3, 5, 7, 9, 11, or 13 with a protein having the amino acidsequence shown in SEQ ID No:2, 4, 6, 8, 10, 12, or 14 being morepreferred. In certain embodiments a MEKK protein is capable of bindingto Rac-GTP. A preferred MEKK protein that is capable of binding to Racor Cdc42 includes a protein encoded by the nucleic acid sequence shownin one of SEQ ID Nos:11 or 13 with a protein having the amino acidsequence represented by one of SEQ ID Nos:12 or 14 being more preferred.

In a preferred embodiment, a MEKK protein of the present invention iscapable of phosphorylating a MEK or MKK, Jun kinase kinase (JNKK) and/orstress activated ERK kinase (SEK), in particular MEK1, MEK2, MKK1, MKK2,MKK3, MKK4, JNKK1, JNKK2, SEK1 and/or SEK2 proteins. As describedherein, MEK1 and MEK2 are equivalent to MKK1 and MKK2, respectively. Inaddition, JNKK1 and JNKK2 are equivalent to MKK3 and MKK4, which areequivalent to SEK1 and SEK2.

A preferred MEKK protein of the present invention is additionallycapable of inducing the phosphorylation of a Myc protein, particularly atranscriptional transactivation domain of Myc, in such a manner that thephosphorylated Myc protein is capable of regulating gene transcription.For example, according to Example 17, a MEKK protein of the presentinvention is preferably capable of inducing luciferase genetranscription by a phosphorylated Myc at least about 25-fold, morepreferably at least about 35-fold, and even more preferably at leastabout 45-fold, over Raf induction when measured under the conditionsdescribed in Example 17.

Another aspect of the present invention relates to the ability of a MEKKactivity to be stimulated by growth factors including, but not limitedto, epidermal growth factor (EGF), neuronal growth factor (NGF), tumornecrosis factor (TNF), C5A, interleukin-8 (IL-8), interleukin-5 (IL-5),monocyte chemotactic protein 1 (MIP1α), monocyte chemoattractant protein1 (MCP-1), platelet activating factor (PAF),N-Formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B₄ (LTB₄R),gastrin releasing peptide (GRP), IgE, major histocompatibility protein(MHC), peptide, superantigen, antigen, vasopressin, thrombin, bradykininand acetylcholine. In addition, the activity of a MEKK protein of thepresent invention is capable of being stimulated by compounds includingphorbol esters such as TPA. A preferred MEKK protein is also capable ofbeing stimulated by EGF, NGF and/or TNF (especially TNFα).

Preferably, the activity of certain of the MEKK proteins of the presentinvention is capable of being stimulated at least 2-fold over basallevels (i.e., levels found in nature when not stimulated), morepreferably at least about 4-fold over basal levels and even morepreferably at least about 6-fold over basal levels, when a cellproducing the MEKK protein is contacted with EGF under the conditionsdescribed in Example 3.

Similarly, the activity of certain of the MEKK proteins of the presentinvention are capable of being stimulated at least 1-fold over basallevels, more preferably at least about 2-fold over basal levels and evenmore preferably at least about 3-fold over basal levels by NGFstimulation, when a cell producing the MEKK protein is contacted withNGF under the conditions described in the appended examples. MEKKproteins which are stimulated by NGF may subsequently cause theactivation of one or more ERKs.

On the other hand, as demonstrated below, certain of the MEKK proteinsof the present invention are capable of being stimulated by removal ofNGF stimulation. MEKK proteins which are stimulated by NGF removal maysubsequently cause the activation of one or more p38 kinases and/orJNKs.

In yet another embodiment, a MEKK protein of the present invention iscapable of being stimulated at least 0.5-fold over basal levels, morepreferably at least about 1-fold over basal levels and even morepreferably at least about 2-fold over basal levels by TPA stimulationwhen a cell producing the MEKK protein is contacted with TPA under theconditions described in Example 9.

TNF is capable of regulating cell death and other functions in differentcell types. Another aspect of the present invention relates to thediscovery that MEKK stimulation by TNF can be independent of Raf.Similarly, the present invention demonstrates that the kinase activityof certain of the subject MEKK proteins can be stimulated by ultravioletlight (UV) damage of cells. It has been observed that both TNF and UVstimulate MEKK activity without substantially activating Raf. Inaddition, both UV and TNF activation of MEKK is apparently Rasdependent. In certain embodiments FGF is capable of preventing TNFinduced apoptosis.

Another aspect of the present invention is the recognition that a MEKKprotein of the present invention is capable of regulating the apoptosisof a cell As used herein, apoptosis refers to the form of cell deaththat comprises: progressive contraction of cell volume with thepreservation of the integrity of cytoplasmic organelles; condensation ofchromatin, as viewed by light or electron microscopy; and DNA cleavage,as electrophoresis or labeling of DNA fragments using terminaldeoxytransferase (TDT). Cell death occurs when the membrane integrity ofthe cell is lost and cell lysis occurs. Apoptosis differs from necrosisin which cells swell and eventually rupture.

A preferred MEKK protein of the present invention is capable of inducingthe apoptosis of cells, such that the cells have characteristicssubstantially similar to cytoplasmic shrinkage and/or nuclearcondensation as described in the appended Examples. The appendedexamples also illustrate that TNF and MEKK can synergize to induceapoptosis in cells.

A schematic representation of an exemplary cell growth regulatory signaltransduction pathway that is MEKK dependent is shown in FIG. 2.Preferred MEKK proteins of the present invention are capable ofregulating the activity of a JNKK protein, JNK protein, Jun proteinand/or ATF-2 protein, and Myc protein, such regulation beingsubstantially, if not entirely, independent of Raf protein. SuchRaf-independent regulation can regulate the growth characteristics of acell, including the apoptosis of a cell. In addition, a MEKK protein ofthe present invention is capable of regulating the activity of MEKprotein, which is also capable of being regulated by Raf protein. Assuch, a MEKK protein of the present invention is capable of regulatingthe activity of MAPK protein and members of the Ets family oftranscription factors, such as TCF protein, also referred to as Elk-1protein.

Referring to FIG. 2, a MEKK protein of the present invention is capableof being activated by a variety of growth factors and environmental cuescapable of activating Ras superfamily protein. In addition, a MEKKprotein is capable of activating JNK protein which is also activated byRas protein, but which is not activated by Raf protein. As such, a MEKKprotein of the present invention comprises a protein kinase at adivergence point in a signal transduction pathway initiated by differentcell surface receptors. A MEKK protein is also capable of beingregulated by TNF protein independent of Raf, thereby indicating anassociation of MEKK protein to a novel signal transduction pathway whichis independent of Ras protein and Raf protein.

Thus, a MEKK protein is capable of performing numerous unique functionsindependent of or by-passing Raf protein in one or more signaltransduction pathways. A MEKK protein is capable of regulating theactivity of MEK and/or JNKK activity. As such, a MEKK protein is capableof regulating the activity of members of a signal transduction pathwaythat does not substantially include Raf activity. Such members include,but are not limited to, JNK, Jun, ATF and Myc protein. In addition, aMEKK protein is capable of regulating the members of a signaltransduction pathway that does involve Raf, such members including, butare not limited to, MEK, MAPK and TCF. A MEKK protein of the presentinvention is thus capable of regulating the apoptosis of a cellindependent of significant involvement by Raf protein.

In addition to the numerous functional characteristics of a MEKKprotein, a MEKK protein of the present invention comprises numerousunique structural characteristics. For example, in one embodiment, aMEKK protein of the present invention includes at least one of twodifferent structural domains having particular functionalcharacteristics. Such structural domains include an NH₂-terminalregulatory domain that serves to regulate a second structural domaincomprising a COOH-terminal protein kinase catalytic domain that iscapable of phosphorylating an MKK protein.

According to the present invention, a MEKK protein of the presentinvention includes a full-length MEKK protein, as well as at least aportion of a MEKK protein capable of performing at least one of thefunctions defined above. The phrase “at least a portion of a MEKKprotein” refers to a portion of a MEKK protein encoded by a nucleic acidmolecule that is capable of hybridizing, under stringent conditions,with a nucleic acid encoding a full-length MEKK protein of the presentinvention. Preferred portions of MEKK proteins are useful for regulatingapoptosis in a cell. Additional preferred portions have activitiesuseful for regulating MEKK kinase activity. Suitable sizes for portionsof a MEKK protein of the present invention are as disclosed for MEKKprotein homologues of the present invention.

In another embodiment, a MEKK protein of the present invention includesat least a portion of a MEKK protein having molecular weights rangingfrom about 70 kD to about 250 kD as determined by Tris-glycine SDS-PAGE,preferably using an 8% polyacrylamide SDS gel (SDS-PAGE) and resolvedusing methods standard in the art. A preferred MEKK protein has amolecular weight ranging from about 65 kD to about 190 kD and even morepreferably from about 69 kD to about 98 kD. In particularly preferredembodiments MEKK2 and MEKK3 proteins of the present invention have amolecular weight of about 65-75 kD. Preferred MEKK4 proteins havemolecular weights about 180-190 kD. Most preferred molecular weights forthe subject MEKKs are: >175 kD (MEKK1), 69.5 kD (MEKK2 or MEKK2.2), 71kD (MEKK3), 185 kD (MEKK4). It is noted that experimental conditionsused when running gels to determine the molecular size of putative MEKKproteins will cause variations in results. Moreover, it has becomeapparent to the Applicant that, relative to predicted molecular weights,shorter apparently related polypeptides can be observed. Whether theseare the result of proteolytic processing, alternative splicing or startcodon usage or the like is unclear, but other preferred MEKK1polypeptides (e.g. MEKK 1.2) have apparent molecular weights of about95-100 kD; and other preferred MEKK4 polypeptides (e.g., MEKK 4.2) haveapparent molecular weights of about 90-100 kD, more preferably 95-98 kD.

In another embodiment, an NH₂-terminal regulatory domain of the presentinvention includes an NH₂-terminal comprising about 400 amino acidshaving at least about 10% serine and/or threonine residues, morepreferably about 400 amino acids having at least about 15% serine and/orthreonine residues, and even more preferably about 400 amino acidshaving at least about 20% serine and/or threonine residues.

In another embodiment an NH₂-terminal regulatory domain of the presentinvention includes an NH₂-terminal comprising about 600 amino acidshaving at least about 10% serine and/or threonine residues, morepreferably about 600 amino acids having at least about 15% serine and/orthreonine residues, and even more preferably about 600 amino acidshaving at least about 20% serine and/or threonine residues.

Another preferred an NH₂-terminal regulatory domain of the presentinvention includes an NH₂-terminal comprising about 1300 amino acidshaving at least about 10% serine and/or threonine residues, morepreferably about 1300 amino acids having at least about 15% serineand/or threonine residues, and even more preferably about 1300 aminoacids having at least about 20% serine and/or threonine residues.

In one embodiment, a MEKK protein of the present invention is devoid ofSH2 and SH3 domains.

A MEKK homologue has at least about 50%, more preferably 75%, morepreferably 85%, and more preferably 95% homology with one of SEQ ID NOs:2, 4, 6, 8, 10, 12, or 14. In preferred embodiments the homolog has 50%,more preferably at least about 75% and more preferably at least about85%, or most preferably at least about 95% amino acid homology with thekinase catalytic domain of a MEKK protein having an amino acid sequencerepresented by one or more of amino acids 409-672 of SEQ ID No:2,1329-1594 of SEQ ID NO:4, amino acids 361-620 of SEQ ID NOs:6 or 8,amino acids 366-626 of SEQ ID NO:10, amino acids, amino acids 631-890 ofSEQ ID NO:12, or amino acids 1338-1597 of SEQ ID NO:14. Anotherpreferred MEKK homologue has at least about 50%, more preferably atleast about 75%, more preferably at least about 85% and even morepreferably about 95% amino acid homology with the NH₂-terminalregulatory domain of a MEKK protein having an amino acid sequencerepresented by amino acids 1-408 of SEQ ID NO:2, amino acids 1-1328 ofSEQ ID NO:4, amino acids 1-360 of SEQ ID NO:6 or 8, amino acids 1-365 ofSEQ ID NO:10, amino acids 1-630 of SEQ ID NO:12, or amino acids 1-1337of SEQ ID No:14.

In another embodiment, a MEKK protein of the present invention includesat least a portion of a MEKK protein homologue preferably has at leastabout 50%, more preferably 75%, more preferably 85%, and more preferably95% homology with one of SEQ ID Nos:2, 4, 6, 8, 10, 12, or 14. In otherembodiments the homolog is 50%, more preferably 75%, more preferably atleast about 85%, and even more preferably at least about 95% amino acidhomology (identity within comparable regions) with the kinase catalyticdomain of a naturally occurring MEKK protein. Another MEKK protein ofthe present invention also includes at least a portion of a MEKKhomologue of the present invention has at least about 50%, morepreferably at least about 75%, or most preferably at least about 85%amino acid homology with the NH₂-terminal regulatory domain of a MEKKprotein of a naturally occurring MEKK protein.

In certain embodiments MEKK proteins have proline rich sequences thatare src homology 3 (SH3) binding motifs. Proline rich regions,specifically the sequence PXXP is thought to be critical in all SH3ligands (Alexandropoulous and Cheng (1995) Proc. Natl. Acad. Sci.92:3110-3114). Preferred MEKK proteins that have proline rich sequencesare encoded by nucleic acids shown in one of SEQ ID Nos:3 or 13. Inparticularly preferred embodiments MEKK proteins comprising SH3 bindingmotifs are shown in one of SEQ ID Nos:4 or 14. Particularly preferredproline rich sequences are exemplified by the sequences shown in aminoacids 26-37 of SEQ ID No:14 or in amino acids 41-51, 70-90, 186-191,211-219 of SEQ ID No:4.

In other embodiments certain MEKK proteins comprise pleckstrin homologydomains. The ‘pleckstrin homology’ (PH) domain is an approximately100-residue protein module that is thought to be involved ininteractions with GTP-binding proteins (Musacchio et al (1993) TIBS28:343-348). Pleckstrin homology domains are very divergent and do notoccupy a specific positions in molecules; alignments of PH domains showsix conserved blocks which all contain several conserved hydrophobicresidues which are thought to form a folded structure comprising sevento eight β-strands, most likely in one or two β-sheets, and just asingle helix (Musacchio et al. supra). PH domains have been identifiedin kinases and also in Vav, Dbl, Bcr, yeast cdc24, Ras-GAP, DM GAP,Ras-GRF, and Sos, all of which are regulators of small GTP-bindingproteins. Interestingly, three of the four proteins that have beenidentified as being capable of binding to SH3 domains (dynamin, 3BP2,and Sos) also contain PH domains (Musacchio et al. supra). The PH domainof β adrenergic receptor kinase may be involved in binding to G proteinβγ complexes (Koch et al. (1993) J. Biol. Chem. 268:8256-8260).Preferred MEKK proteins that have PH domains are encoded by nucleicacids shown in one of SEQ ID Nos:3 or 13. In particularly preferredembodiments MEKK proteins comprising PH domains are shown in one of SEQID Nos:4 or 14. Particularly preferred PH domains are exemplified by theamino acids 262-665 of SEQ ID No:4 or amino acids 233-397 of SEQ IDNo:14.

In another embodiment the MEKK proteins of the present invention bind toMKK substrates. Preferred MEKK proteins comprise consensus MKK bindingdomains as encoded by the nucleic acid sequences shown in one of SEQ IDNos: 1, 3, 5, 7, 9, 11, or 13. Preferred MKK consensus binding regionsare illustrated by amino acids 658-672 of SEQ ID No:2, amino acids1579-1593 of SEQ ID No:4, amino acids 605-620 of SEQ ID Nos: 6 or 8,amino acids 611-626 of SEQ ID No:10, amino acids 872-890 of SEQ IDNo:12, or amino acids 1579-1597 of SEQ ID No:14.

The sequences comprising the catalytic domain of a MEKK protein areinvolved in phosphotransferase activity, and therefore display arelatively conserved amino acid sequence. The NH₂-terminal regulatorydomain of a MEKK protein, however, can be substantially divergent. Thelack of significant homology between MEKK protein NH₂-terminalregulatory domains is related to the regulation of each of such domainsby different upstream regulatory proteins. For example, a MEKK proteincan be regulated by the protein Ras, while others can be regulatedindependent of Ras. In addition, some MEKK proteins can be regulated bythe growth factor TNFα, while others cannot. As such, the NH₂-terminalregulatory domain of a MEKK protein provides selectivity for upstreamsignal transduction regulation, while the catalytic domain provides forMEKK substrate selectivity function.

In a preferred embodiment, a MEKK protein of the present inventionincludes at least a portion of a MEKK protein homologue of the presentinvention that is encoded by a nucleic acid molecule preferably has atleast about 50%, more preferably 75%, more preferably 85%, and morepreferably 95% homology with one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, or14. Preferred fragments of MEKK proteins include those in which at leasta portion of a MEKK regulatory domain is deleted to form aconstitutively active molecule, or those in which at least a portion ofa MEKK catalytic domain is deleted to form a catalyticly inactivemolecule.

Still another preferred MEKK homologue is encoded by a nucleic acidmolecule having at least about 50%, more preferably 75%, more preferably85%, and more preferably 95% homology with one of SEQ ID Nos:1, 3, 5, 7,9, 11, or 13. In other embodiments the nucleic acid has at least about50%, more preferably at least about 75%, more preferably at least about85%, or most preferably at least about 95% homologous with the kinasecatalytic domain of a MEKK protein encoded by a nucleic acid sequencerepresented by SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13. A MEKK homologuealso includes those encoded by a nucleic acid molecule having at leastabout 50%, more preferably at least about 75%, more preferably at leastabout 85%, and even more preferably at least about 95% amino acidhomology with the NH₂-terminal regulatory domain of a MEKK proteinencoded by a nucleic acid sequence represented by SEQ ID Nos: 1, 3, 5,7, 9, 11, or 13.

In another embodiment, the subject MEKK proteins are provided as fusionproteins. It is widely appreciated that fusion proteins can alsofacilitate the expression of proteins, and accordingly, can be used inthe expression of the MEKK polypeptides of the present invention. Forexample, MEKK polypeptides can be generated as glutathione-S-transferase(GST-fusion) proteins. Such GST-fusion proteins can enable easypurification of the MEKK polypeptide, as for example by the use ofglutathione-derivatized matrices (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant protein, canallow purification of the expressed fusion protein by affinitychromatography using a Ni2+ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinaseto provide the purified protein (e.g., see Hochuli et al. (1987) J.Chromatography 411:177; and Janknecht et al. PNAS 88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

According to the present invention, a MEKK protein of the presentinvention can include MEKK proteins that have undergonepost-translational modification. Such modification can include, forexample, phosphorylation or among other post-translational modificationsincluding conformational changes or post-translational deletions.

This invention further contemplates a method for generating sets ofcombinatorial mutants of the subject MEKK proteins as well as truncationmutants, and is especially useful for identifying potential variantsequences (e.g. homologs) that are functional in modulating signaltransduction. The purpose of screening such combinatorial libraries isto generate, for example, novel MEKK homologs which can act as eitheragonists or antagonist of the wild-type MEKK proteins, or alternatively,which possess novel activities all together. To illustrate, MEKKhomologs can be engineered by the present method to provide selective,constitutive activation of a pathway, so as mimic induction by a factorwhen the MEKK homolog is expressed in a cell capable of responding tothe factor. Thus, combinatorially-derived homologs can be generated tohave an increased potency relative to a naturally occurring form of theprotein.

Likewise, MEKK homologs can be generated by the present combinatorialapproach to selectively inhibit (antagonize) induction by a growth orother factor. For instance, mutagenesis can provide MEKK homologs whichare able to bind other signal pathway proteins (e.g., MEKs) yet preventpropagation of the signal, e.g. the homologs can be dominant negativemutants. Moreover, manipulation of certain domains of MEKK by thepresent method can provide domains more suitable for use in fusionproteins.

In one aspect of this method, the amino acid sequences for a populationof MEKK homologs or other related proteins are aligned, preferably topromote the highest homology possible. Such a population of variants caninclude, for example, MEKK homologs from one or more species. Aminoacids which appear at each position of the aligned sequences areselected to create a degenerate set of combinatorial sequences. In apreferred embodiment, the variegated library of MEKK variants isgenerated by combinatorial mutagenesis at the nucleic acid level, and isencoded by a variegated gene library. For instance, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential MEKK sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g. for phage display) containing the set ofMEKK sequences therein.

There are many ways by which such libraries of potential MEKK homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential MEKK sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386-390;Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Likewise, a library of coding sequence fragments can be provided for aMEKK clone in order to generate a variegated population of MEKKfragments for screening and subsequent selection of bioactive fragments.A variety of techniques are known in the art for generating suchlibraries, including chemical synthesis. In one embodiment, a library ofcoding sequence fragments can be generated by (i) treating a doublestranded PCR fragment of a MEKK coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule; (ii)denaturing the double stranded DNA; (iii) renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products; (iv) removing single stranded portions fromreformed duplexes by treatment with S1 nuclease; and (v) ligating theresulting fragment library into an expression vector. By this exemplarymethod, an expression library can be derived which codes for N-terminal,C-terminal and internal fragments of various sizes.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of MEKK homologs. The most widely used techniques forscreening large gene libraries typically comprise cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate MEKK sequences created bycombinatorial mutagenesis techniques.

In an illustrative embodiment of a screening assay, the gene library canbe expressed as a fusion protein on the surface of a viral particle. Forinstance, in the filamentous phage system, foreign peptide sequences canbe expressed on the surface of infectious phage, thereby conferring twosignificant benefits. First, since these phage can be applied toaffinity matrices at very high concentrations, a large number of phagecan be screened at one time. Second, since each infectious phagedisplays the combinatorial gene product on its surface, if a particularphage is recovered from an affinity matrix in low yield, the phage canbe amplified by another round of infection. The group of almostidentical E. coli filamentous phages M13, fd, and fl are most often usedin phage display libraries, as either of the phage gIII or gVIII coatproteins can be used to generate fusion proteins without disrupting theultimate packaging of the viral particle (Ladner et al. PCT publicationWO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al.(1992) J. Biol. Chem. 267:16007-16010; Griffths et al. (1993) EMBO J12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al.(1992) PNAS 89:4457-4461). The resulting phage libraries with the fusiontail proteins may be panned, e.g. using a fluorescently labeled MEKprotein, e.g. FITC-MEK, to score for MEKK homologs which retain theability to bind to the MEK protein. Individual phage which encode a MEKKhomolog which retains MEK binding can be isolated, the MEKK homolog generecovered from the isolate, and further tested to discern between activeand antagonistic mutants

In another embodiment, the REF52 cells of Example 18 or 19 can beexploited to analyze the variegated MEKK library. For instance, thelibrary of expression vectors can be transfected into a population ofREF52 cells which also inducibly overexpress a MEKK protein (e.g., andwhich overexpression causes apoptosis). Expression of WT-MEKK is theninduced. and the effect of the MEKK mutant on induction of apoptosis canbe detected. Plasmid DNA can then be recovered from the cells whichscore for inhibition, or alternatively, potentiation of apoptosis, andthe individual clones further characterized.

The invention also provides for reduction of the MEKK proteins togenerate mimetics, e.g. peptide or non-peptide agents, which are able todisrupt binding of a MEKK polypeptide of the present invention witheither upstream or downstream components of its signaling cascade. Thus,such mutagenic techniques as described above are also useful to map thedeterminants of the MEKK proteins which participate in protein-proteininteractions involved in, for example, binding of the subject MEKKpolypeptide to proteins which may function upstream (including bothactivators and repressors of its activity) or to proteins which mayfunction downstream of the MEKK polypeptide, whether they are positivelyor negatively regulated by it. To illustrate, the critical residues of asubject MEKK polypeptide which are involved in molecular recognition ofan upstream or downstream MEKK component can be determined and used togenerate MEKK-derived peptidomimetics which competitively inhibitbinding of the authentic protein with that moiety. By employing, forexample, scanning mutagenesis to map the amino acid residues of each ofthe subject MEKK proteins which are involved in binding other cellularproteins, peptidomimetic compounds can be generated which mimic thoseresidues of the MEKK protein which facilitate the interaction. Suchmimetics may then be used to interfere with the normal function of aMEKK protein. For instance, non-hydrolyzable peptide analogs of suchresidues can be generated using benzodiazepine (e.g., see Freidinger etal. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOMPublisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al.in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey etal. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOMPublisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides(Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. inPeptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), and β-aminoalcohols(Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann etal. (1986) Biochem Biophys Res Commun 134:71).

Another aspect of the present invention is an isolated nucleic acidmolecule capable of hybridizing, under stringent conditions, with a MEKKprotein gene encoding a MEKK protein of the present invention. Inaccordance with the present invention, an isolated nucleic acid moleculeis a nucleic acid molecule that has been removed from its natural milieu(i.e., that has been subject to human manipulation). As such, “isolated”does not reflect the extent to which the nucleic acid molecule has beenpurified. To this end, the term “isolated” as used herein with respectto nucleic acids, such as DNA or RNA, refers to molecules separated fromother DNAs, or RNAs, respectively, that are present in the naturalsource of the macromolecule. For example, an isolated nucleic acidencoding one of the subject MEKK polypeptides preferably includes nomore than 10 kilobases (kb) of nucleic acid sequence which naturallyimmediately flanks the MEKK gene in genomic DNA, more preferably no morethan 5 kb of such naturally occurring flanking sequences, and mostpreferably less than 1.5 kb of such naturally occurring flankingsequence. The term isolated as used herein will also be understood toinclude nucleic acid that is substantially free of cellular material,viral material, or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. Moreover, an “isolated nucleic acid” is meant to includenucleic acid fragments which are not naturally occurring as fragmentsand would not be found in the natural state.

An isolated nucleic acid molecule can include DNA, RNA, or derivativesof either DNA or RNA. Accordingly, as used herein, the term “nucleicacid” includes polynucleotides such as deoxyribonucleic acid (DNA), and,where appropriate, ribonucleic acid (RNA). The term should also beunderstood to include, as equivalents, analogs of either RNA or DNA madefrom nucleotide analogs, and, as applicable to the embodiment beingdescribed, single (sense or antisense) and double-strandedpolynucleotides.

As used herein, the term “gene” or “recombinant gene” includes nucleicacid comprising an open reading frame encoding one of the MEKKpolypeptides of the present invention, including both exon and(optionally) intron sequences. A “recombinant gene” refers to nucleicacid encoding a MEKK polypeptide and comprising MEKK-encoding exonsequences, though it may optionally include intron sequences which areeither derived from a chromosomal MEKK gene or from an unrelatedchromosomal gene. Exemplary recombinant genes encoding the subject MEKKpolypeptides are represented in the appended Sequence Listing.

An isolated nucleic acid molecule of the present invention can beobtained from its natural source either as an entire (i.e., complete)gene or a portion thereof capable of forming a stable hybrid with thatgene. As used herein, the phrase “at least a portion of” an entityrefers to an amount of the entity that is at least sufficient to havethe functional aspects of that entity. For example, at least a portionof a nucleic acid sequence, as used herein, is an amount of a nucleicacid sequence capable of forming a stable hybrid with a particulardesired gene (e.g., MEKK genes) under stringent hybridizationconditions. An isolated nucleic acid molecule of the present inventioncan also be produced using recombinant DNA technology (e.g., polymerasechain reaction (PCR) amplification, cloning) or chemical synthesis.Isolated MEKK protein nucleic acid molecules include natural nucleicacid molecules and homologues thereof, including, but not limited to,natural allelic variants and modified nucleic acid molecules in whichnucleotides have been inserted, deleted, substituted, and/or inverted insuch a manner that such modifications do not substantially interferewith the nucleic acid molecule's ability to encode a MEKK protein of thepresent invention or to form stable hybrids under stringent conditionswith natural nucleic acid molecule isolates of MEKK.

Preferred modifications to a MEKK protein nucleic acid molecule of thepresent invention include truncating a full-length MEKK protein nucleicacid molecule by, for example: deleting at least a portion of a MEKKprotein nucleic acid molecule encoding a regulatory domain to produce aconstitutively active MEKK protein; deleting at least a portion of aMEKK protein nucleic acid molecule encoding a catalytic domain toproduce an inactive MEKK protein; and modifying the MEKK protein toachieve desired inactivation and/or stimulation of the protein, forexample, substituting a codon encoding a lysine residue in the catalyticdomain (i.e., phosphotransferase domain) with a methionine residue toinactivate the catalytic domain.

A preferred truncated MEKK nucleic acid molecule encodes a form of aMEKK protein containing a catalytic domain but that lacks a regulatorydomain. Preferred catalytic domain truncated MEKK nucleic acid moleculesencode amino acid residues from about 409 to about 672 of MEKK 1.1;amino acids 1331 to about 1594 of MEKK 1.2; from about 361 to about 620of MEKK 2.1 or 2.2; from about 366 to about 626 of MEKK 3; from about631 to about 890 of MEKK4.1; or from about 1338 to about 1597 for MEKK4.2.

Another preferred truncated MEKK nucleic acid molecule encodes a form ofa MEKK protein comprising an NH₂-terminal regulatory domain a catalyticdomain but lacking a catalytic domain. Preferred regulatory domaintruncated MEKK nucleic acid molecules encode amino acid residues fromabout 1 to about 408 of MEKK 1.1; amino acids 1 to about 1328 of MEKK1.2; from about 1 to about 360 of MEKK 2.1 or 2.2; from about 1 to about365 of MEKK 3; from about 1 to about 630 of MEKK 4.1; or from about 1 toabout 1337 for MEKK 4.2.

An isolated nucleic acid molecule of the present invention can include anucleic acid sequence that encodes at least one MEKK protein of thepresent invention, examples of such proteins being disclosed herein.Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”primarily refers to the sequence of nucleotides that comprise thenucleic acid molecule, the two phrases can be used interchangeably. Asheretofore disclosed, MEKK proteins of the present invention include,but are not limited to, proteins having full-length MEKK protein codingregions, portions thereof, and other MEKK protein homologues.

As used herein, a MEKK protein gene includes all nucleic acid sequencesrelated to a natural MEKK protein gene such as regulatory regions thatcontrol production of a MEKK protein encoded by that gene (including,but not limited to, transcription, translation or post-translationcontrol regions) as well as the coding region itself. A nucleic acidmolecule of the present invention can be an isolated natural MEKKprotein nucleic acid molecule or a homologue thereof. A nucleic acidmolecule of the present invention can include one or more regulatoryregions, full-length or partial coding regions, or combinations thereof.The minimal size of a MEKK protein nucleic acid molecule of the presentinvention is the minimal size capable of forming a stable hybrid understringent hybridization conditions with a corresponding natural gene.

A MEKK protein nucleic acid molecule homologue can be produced using anumber of methods known to those skilled in the art (see, e.g., Sambrooket al., ibid.). For example, nucleic acid molecules can be modifiedusing a variety of techniques including, but not limited to, classicmutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, polymerase chain reaction(PCR) amplification and/or mutagenesis of selected regions of a nucleicacid sequence, synthesis of oligonucleotide mixtures and ligation ofmixture groups to “build” a mixture of nucleic acid molecules andcombinations thereof. Nucleic acid molecule homologues can be selectedfrom a mixture of modified nucleic acids by screening for the functionof the protein encoded by the nucleic acid (e.g., the ability of ahomologue to phosphorylate MEK protein or JNKK protein) and/or byhybridization with isolated MEKK protein nucleic acids under stringentconditions.

One embodiment of the present invention is a MEKK protein nucleic acidmolecule capable of encoding at least a portion of a MEKK protein, or ahomologue thereof, as described herein. A preferred nucleic acidmolecule of the present invention includes, but is not limited to, anucleic acid molecule that encodes a protein having at least a portionof an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14, orhomologues thereof. Proteins at least 50%, preferably at least about75%, more preferably at least about 85%, and most preferably at leastabout 95% homologous to these sequences are contemplated.

A preferred nucleic acid molecule of the present invention is capable ofhybridizing under stringent conditions to a nucleic acid that encodes atleast a portion of a MEKK protein, or a homologue thereof. Alsopreferred is a MEKK protein nucleic acid molecule that includes anucleic acid sequence having at least about 50% homology, preferably 75%homology, preferably 85% homology, or even more preferably 95% homologywith one of SEQ ID No:1, 3, 5, 7, 9, 11, or 13. In other embodimentsnucleic acids have 50%, preferably at least about 75%, and morepreferably at least about 85%, and most preferably at least about 95%homology with the corresponding region(s) of the nucleic acid sequenceencoding the catalytic domain of a MEKK protein, or a homologue thereof.Also preferred is a MEKK protein nucleic acid molecule that includes anucleic acid sequence having at least about 50%, preferably at leastabout 75%, more preferably at least about 85%, and even more preferablyat least about 95% homology with the corresponding region(s) of thenucleic acid sequence encoding the NH₂-terminal regulatory domain of aMEKK protein, or a homologue thereof. A particularly preferred nucleicacid sequence is a nucleic acid sequence having at least about 50%,preferably at least about 75%, and more preferably at least about 85%,and most preferably at least about 95% homology with a nucleic acidsequence encoding the catalytic domain amino acid residues from about409 to about 672 of SEQ ID No:2; amino acids 1331 to about 1594 of SEQID No:4; from about 361 to about 620 of SEQ ID No:6 or 8; from about 366to about 626 of SEQ ID No:10; from about 631 to about 890 of SEQ IDNo:12; or from about 1338 to about 1597 for SEQ ID No:14. Anotherpreferred MEKK homologue has at least about 50%, more preferably atleast about 75%, more preferably at least about 85% and even morepreferably about 95% amino acid homology with the NH₂-terminalregulatory domain of a MEKK protein having an amino acid sequencerepresented by amino acid residues from about 1 to about 408 of SEQ IDNo:2; amino acids 1 to about 1328 of SEQ ID No:4; from about 1 to about360 of SEQ ID No:6 or 8; from about 1 to about 365 of SEQ ID No:10; fromabout 1 to about 630 of SEQ ID No:12; or from about 1 to about 1337 forSEQ ID No:14.

Such nucleic acid molecules can be a full-length gene and/or a nucleicacid molecule encoding a full-length protein, a hybrid protein, a fusionprotein, a multivalent protein or a truncation fragment. More preferrednucleic acid molecules of the present invention comprise isolatednucleic acid molecules having a nucleic acid sequence as represented byone of SEQ ID Nos: 1, 3, 5, 7, 9, 11, or 13, or nucleic acid moleculescapable of hybridizing to said sequences under stringent conditions.

Knowing a nucleic acid molecule of a MEKK protein of the presentinvention allows one skilled in the art to make copies of that nucleicacid molecule as well as to obtain additional portions of MEKKprotein-encoding genes (e.g., nucleic acid molecules that include thetranslation start site and/or transcription and/or translation controlregions), and/or MEKK protein nucleic acid molecule homologues. Knowinga portion of an amino acid sequence of a MEKK protein of the presentinvention allows one skilled in the art to clone nucleic acid sequencesencoding such a MEKK protein.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent conditions,with complementary regions of other, preferably longer, nucleic acidmolecules of the present invention that encode at least a portion of aMEKK protein, or a homologue thereof. A preferred oligonucleotide iscapable of hybridizing, under stringent conditions, with a nucleic acidmolecule that is capable of encoding at least a portion of an amino acidsequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID No:14, or homologues thereof. Amore preferred oligonucleotide is capable of hybridizing to a nucleicacid molecule having a nucleic acid sequence as represented by SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID No:13 or complements thereof.

Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimal size of such oligonucleotides is thesize required to form a stable hybrid between a given oligonucleotideand the complementary sequence on another nucleic acid molecule of thepresent invention. Minimal size characteristics of preferredoligonucleotides are at least about 10 nucleotides, preferably at leastabout 20 nucleotides, more preferably at least about 50 nucleotides andmost preferably at least about 60 nucleotides. Larger fragments are alsocontemplated. The size of the oligonucleotide must also be sufficientfor the use of the oligonucleotide in accordance with the presentinvention. Oligonucleotides of the present invention can be used in avariety of applications including, but not limited to, as probes toidentify additional nucleic acid molecules, as primers to amplify orextend nucleic acid molecules or in therapeutic applications to inhibit,for example, expression of MEKK proteins by cells. Such therapeuticapplications include the use of such oligonucleotides in, for example,antisense-, triplex formation-, ribozyme- and/or RNA drug-basedtechnologies. The present invention, therefore, includes use of sucholigonucleotides and methods to interfere with the production of MEKKproteins. In addition oligonucleotides encoding portions of MEKKproteins which bind to MEKK binding proteins can be used a therapeutics.In other embodiments, the peptides encoded by these nucleic acids areused.

To further illustrate, another aspect of the invention relates to theuse of the isolated nucleic acid in “antisense” therapy. As used herein,“antisense” therapy refers to administration or in situ generation ofoligonucleotide probes or their derivatives which specifically hybridize(e.g. bind) under cellular conditions, with the cellular mRNA and/orgenomic DNA encoding one or more of the subject MEKK proteins so as toinhibit expression of that protein, e.g. by inhibiting transcriptionand/or translation. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes a vertebrate MEKK protein. Alternatively,the antisense construct is an oligonucleotide probe which is generatedex vivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of avertebrate MEKK gene. Such oligonucleotide probes are preferablymodified oligonucleotides which are resistant to endogenous nucleases,e.g. exonucleases and/or endonucleases, and are therefore stable invivo. Exemplary nucleic acid molecules for use as antisenseoligonucleotides are phosphoramidate, phosphothioate andmethylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996;5,264,564; and 5,256,775). Additionally, general approaches toconstructing oligomers useful in antisense therapy have been reviewed,for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; andStein et al. (1988) Cancer Res 48:2659-2668.

Accordingly, the modified oligomers of the invention are useful intherapeutic, diagnostic, and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general. For such therapy, the oligomers of theinvention can be formulated for a variety of loads of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. For systemic administration,injection is preferred, including intramuscular, intravenous,intraperitoneal, and subcutaneous. For injection, the oligomers of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligomers may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms arealso included.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration bile salts and fusidic acid derivatives. In addition,detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays or using suppositories. Fororal administration, the oligomers are formulated into conventional oraladministration forms such as capsules, tablets, and tonics. For topicaladministration, the oligomers of the invention are formulated intoointments, salves, gels, or creams as generally known in the art.

In addition to use in therapy, the oligomers of the invention may beused as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind. Suchdiagnostic tests are described in further detail below.

Likewise, the antisense constructs of the present invention, byantagonizing the normal biological activity of one of the MEKK proteins,can be used in the manipulation of tissue, e.g. tissue differentiation,both in vivo and for ex vivo tissue cultures.

Furthermore, the anti-sense techniques (e.g. microinjection of antisensemolecules, or transfection with plasmids whose transcripts areanti-sense with regard to a MEKK mRNA or gene sequence) can be used toinvestigate role of MEKK in disease states, as well as the normalcellular function of MEKK in healthy tissue. Such techniques can beutilized in cell culture, but can also be used in the creation oftransgenic animals. The present invention also includes a recombinantvector which includes at least one MEKK protein nucleic acid molecule ofthe present invention inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, for example nucleic acid sequencesthat are not naturally found adjacent to MEKK protein nucleic acidmolecules of the present invention. The vector can be either RNA or DNA,and either prokaryotic or eukaryotic, and is typically a virus or aplasmid. Recombinant vectors can be used in the cloning, sequencing,and/or otherwise manipulating of MEKK protein nucleic acid molecules ofthe present invention. One type of recombinant vector, herein referredto as a recombinant molecule and described in more detail below, can beused in the expression of nucleic acid molecules of the presentinvention. Preferred recombinant vectors are capable of replicating inthe transformed cell.

Preferred nucleic acid molecules to insert into a recombinant vectorincludes a nucleic acid molecule that encodes at least a portion of aMEKK protein, or a homologue thereof. A more preferred nucleic acidmolecule to insert into a recombinant vector includes a nucleic acidmolecule encoding at least a portion of an amino acid sequencerepresented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, and/or SEQ ID No:14, or homologues thereof. Aneven more preferred nucleic acid molecule to insert into a recombinantvector includes a nucleic acid molecule represented by SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, and/or SEQID No:13 or complements thereof. In particularly preferred embodimentsportions of a MEKK nucleic acid which encodes a MEKK catalytic domain isused. In another particularly preferred embodiment, at least a portionof a nucleic acid which encodes the portion of a MEKK protein whichbinds to a MEKK substrate or a MEKK regulatory protein is used.

Suitable host cells for transforming a cell can include any cell capableof producing MEKK proteins of the present invention after beingtransformed with at least one nucleic acid molecule of the presentinvention. Host cells can be either untransformed cells or cells thatare already transformed with at least one nucleic acid molecule.Suitable host cells of the present invention can include bacterial,fungal (including yeast), insect, animal and plant cells. Preferred hostcells include bacterial, yeast, insect and mammalian cells, withmammalian cells being particularly preferred.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase operatively linked refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.As used herein, an expression vector is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of aspecified nucleic acid molecule. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors canbe either prokaryotic or eukaryotic, and are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in recombinantcells of the present invention, including in bacterial, fungal, insect,animal, and/or plant cells. As such, nucleic acid molecules of thepresent invention can be operatively linked to expression vectorscontaining regulatory sequences such as promoters, operators,repressors, enhancers, termination sequences, origins of replication,and other regulatory sequences that are compatible with the recombinantcell and that control the expression of nucleic acid molecules of thepresent invention. As used herein, a transcription control sequenceincludes a sequence which is capable of controlling the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those which control transcriptioninitiation, such as promoter, enhancer, operator and repressorsequences. Suitable transcription control sequences include anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin bacterial, yeast, and mammalian cells, such as, but not limited to,tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (λ)(such as λp_(L) and λp_(R) and fusions that include such promoters),bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6,bacteriophage SP01, metallothionein, alpha mating factor, baculovirus,vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40,retrovirus actin, retroviral long terminal repeat, Rous sarcoma virus,heat shock, phosphate and nitrate transcription control sequences, aswell as other sequences capable of controlling gene expression inprokaryotic or eukaryotic cells. Additional suitable transcriptioncontrol sequences include tissue-specific promoters and enhancers aswell as lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins). Transcription control sequences of thepresent invention can also include naturally occurring transcriptioncontrol sequences naturally associated with a DNA sequence encoding aMEKK protein.

Preferred nucleic acid molecules for insertion into an expression vectorinclude nucleic acid molecules that encode at least a portion of a MEKKprotein, or a homologue thereof. A more preferred nucleic acid moleculefor insertion into an expression vector includes a nucleic acid moleculeencoding at least a portion of an amino acid sequence represented by SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ BD NO:8, SEQ ID NO:10, SEQ IDNO:12, and/or SEQ ID No:14 or homologues thereof. An even more preferrednucleic acid molecule for insertion into an expression vector includes anucleic acid molecule represented by SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 and/or SEQ ID No:13, orcomplements thereof.

Expression vectors of the present invention may also contain fusionsequences which lead to the expression of inserted nucleic acidmolecules of the present invention as fusion proteins. Inclusion of afusion sequence as part of a MEKK nucleic acid molecule of the presentinvention can enhance the stability during production, storage and/oruse of the protein encoded by the nucleic acid molecule. Furthermore, afusion segment can function as a tool to simplify purification of a MEKKprotein, such as to enable purification of the resultant fusion proteinusing affinity chromatography. A suitable fusion segment can be a domainof any size that has the desired function (e.g., increased stabilityand/or purification tool). It is within the scope of the presentinvention to use one or more fusion segments. Fusion segments can bejoined to amino and/or carboxyl termini of a MEKK protein. Linkagesbetween fusion segments and MEKK proteins can be constructed to besusceptible to cleavage to enable straight-forward recovery of the MEKKproteins. Fusion proteins are preferably produced by culturing arecombinant cell transformed with a fusion nucleic acid sequence thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end of a MEKK protein.

Moreover, the gene constructs of the present invention can also be usedas a part of a gene therapy protocol to deliver nucleic acids encodingeither an agonistic or antagonistic form of one of the subject MEKKproteins. Thus, another aspect of the invention features expressionvectors for in vivo or in vitro transfection and expression of a MEKKpolypeptide in particular cell types so as to reconstitute the functionof, constitutively activate, or alternatively, abrogate the function ofa signal pathway dependent on a MEKK activity. Such therapies may usefulwhere the naturally-occurring form of the protein is misexpressed orinappropriately activated; or to deliver a form of the protein whichalters differentiation of tissue; or which inhibits neoplastictransformation.

Expression constructs of the subject MEKK polypeptide, and mutantsthereof, may be administered in any biologically effective carrier, e.g.any formulation or composition capable of effectively delivering therecombinant gene to cells in vivo. Approaches include insertion of thesubject gene in viral vectors including recombinant retroviruses,adenovirus, adeno-associated virus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors transfectcells directly; plasmid DNA can be delivered with the help of, forexample, cationic liposomes (lipofectin) or derivatized (e.g. antibodyconjugated), polylysine conjugates, gramacidin S, artificial viralenvelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation carried out invivo. It will be appreciated that because transduction of appropriatetarget cells represents the critical first step in gene therapy, choiceof the particular gene delivery system will depend on such factors asthe phenotype of the intended target and the route of administration,e.g. locally or systemically. Furthermore, it will be recognized thatthe particular gene construct provided for in vivo transduction of MEKKexpression are also useful for in vitro transduction of cells, such asfor use in the ex vivo tissue culture systems described below.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNA,encoding the particular MEKK polypeptide desired. Infection of cellswith a viral vector has the advantage that a large proportion of thetargeted cells can receive the nucleic acid. Additionally, moleculesencoded within the viral vector, e.g., by a cDNA contained in the viralvector, are expressed efficiently in cells which have taken up viralvector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termed “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding oneof the subject proteins rendering the retrovirus replication defective.The replication defective retrovirus is then packaged into virions whichcan be used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are well known to those skilled inthe art. Examples of suitable packaging virus lines for preparing bothecotropic and amphotropic retroviral systems include ψCrip, ψCre, ω2 andψAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including neuronal cells, in vitro and/or invivo (see for example Eglitis, et al. (1985) Science 230:1395-1398;Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464;Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentanoet al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al.(1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991)Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al.(1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J.Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCTApplication WO 89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J BiolChem 266:14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (e.g. lactose to convertthe env protein to an asialoglycoprotein), as well as by generatingfusion proteins (e.g. single-chain antibody/env fusion proteins). Thistechnique, while useful to limit or otherwise direct the infection tocertain tissue types, can also be used to convert an ecotropic vector into an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequenceswhich control expression of the MEKK gene of the retroviral vector.

Another viral gene delivery system useful in the present inventionutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Berkner et al. (1988)Biotechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses can be advantageous in certaincircumstances in that they can be used to infect a wide variety of celltypes, including airway epithelium (Rosenfeld et al. (1992) citedsupra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad.Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl.Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992)Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the virusparticle is relatively stable and amenable to purification andconcentration, and as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situations whereintroduced DNA becomes integrated into the host genome (e.g., retroviralDNA). Moreover, the carrying capacity of the adenoviral genome forforeign DNA is large (up to 8 kilobases) relative to other gene deliveryvectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J.Virol. 57:267). Most replication-defective adenoviral vectors currentlyin use and therefore favored by the present invention are deleted forall or parts of the viral E1 and E3 genes but retain as much as 80% ofthe adenoviral genetic material (see, e.g., Jones et al. (1979) Cell16:683; Berkner et al., supra; and Graham et al. in Methods in MolecularBiology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp.109-127). Expression of the inserted MEKK gene can be under control of,for example, the E1A promoter, the major late promoter (MLP) andassociated leader sequences, the E3 promoter, or exogenously addedpromoter sequences.

Yet another viral vector system useful for delivery of one of thesubject MEKK genes is the adeno-associated virus (AMINO ACIDSV).Adeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle. (Fora review see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)158:97-129). It is also one of the few viruses that may integrate itsDNA into non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al. (1992) Am. J. Respir. Cell.Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; andMcLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing aslittle as 300 base pairs of AMINO ACIDSV can be packaged and canintegrate. Space for exogenous DNA is limited to about 4.5 kb. An AMINOACIDSV vector such as that described in Tratschin et al. (1985) Mol.Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. Avariety of nucleic acids have been introduced into different cell typesusing AMINO ACIDSV vectors (see for example Hermonat et al. (1984) Proc.Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell.Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39;Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993)J. Biol. Chem. 268:3781-3790).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a subjectMEKK polypeptide in the tissue of an animal. Most nonviral methods ofgene transfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the subject MEKKpolypeptide gene by the targeted cell. Exemplary gene delivery systemsof this type include liposomal derived systems, poly-lysine conjugates,and artificial viral envelopes.

In clinical settings, the gene delivery systems for the therapeutic MEKKgene can be introduced into a patient by any of a number of methods,each of which is familiar in the art. For instance, a pharmaceuticalpreparation of the gene delivery system can be introduced systemically,e.g. by intravenous injection, and specific transduction of the proteinin the target cells occurs predominantly from specificity oftransfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054-3057). A MEKK gene, such as any one of the clonesrepresented in the appended Sequence Listing, can be delivered in a genetherapy construct by electroporation using techniques described, forexample, by Dev et al. ((1994) Cancer Treat Rev 20:105-115).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

Still another aspect of the present invention pertains to recombinantcells, e.g., cells which are transformed with at least one of anynucleic acid molecule of the present invention. A preferred recombinantcell is a cell transformed with at least one nucleic acid molecule thatencodes at least a portion of a MEKK protein, or a homologue thereof. Amore preferred recombinant cell is transformed with at least one nucleicacid molecule that is capable of encoding at least a portion of an aminoacid sequence represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12 and/or SEQ ID No:14, or homologuesthereof. An even more preferred recombinant cell is transformed with atleast one nucleic acid molecule represented by SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, and/or SEQ IDNo:13, or complements thereof. Particularly preferred recombinant cellsinclude mammalian cells involved in a disease transformed with at leastone of the aforementioned nucleic acid molecules.

It may be appreciated by one skilled in the art that use of recombinantDNA technologies can improve expression of transformed nucleic acidmolecules by manipulating, for example, the number of copies of thenucleic acid molecules within a host cell, the efficiency with whichthose nucleic acid molecules are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful forincreasing the expression of nucleic acid molecules of the presentinvention include, but are not limited to, operatively linking nucleicacid molecules to high-copy number plasmids, integration of the nucleicacid molecules into one or more host cell chromosomes, addition ofvector stability sequences to plasmids, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgamo sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant protein productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing the resultant protein.

As used herein, amplifying the copy number of a nucleic acid sequence ina cell can be accomplished either by increasing the copy number of thenucleic acid sequence in the cell's genome or by introducing additionalcopies of the nucleic acid sequence into the cell by transformation.Copy number amplification is conducted in a manner such that greateramounts of enzyme are produced, leading to enhanced conversion ofsubstrate to product. For example, recombinant molecules containingnucleic acids of the present invention can be transformed into cells toenhance enzyme synthesis. Transformation can be accomplished using anyprocess by which nucleic acid sequences are inserted into a cell. Priorto transformation, the nucleic acid sequence on the recombinant moleculecan be manipulated to encode an enzyme having a higher specificactivity.

In accordance with the present invention, recombinant cells can be usedto produce a MEKK protein of the present invention by culturing suchcells under conditions effective to produce such a protein, andrecovering the protein. Effective conditions to produce a proteininclude, but are not limited to, appropriate media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Anappropriate, or effective, medium refers to any medium in which a cellof the present invention, when cultured, is capable of producing a MEKKprotein. Such a medium is typically an aqueous medium comprisingassimilable carbohydrate, nitrogen and phosphate sources, as well asappropriate salts, minerals, metals and other nutrients, such asvitamins. The medium may comprise complex nutrients or may be a definedminimal medium.

A preferred cell to culture is a recombinant cell that is capable ofexpressing the MEKK protein, the recombinant cell being produced bytransforming a host cell with one or more nucleic acid molecules of thepresent invention. Transformation of a nucleic acid molecule into a cellcan be accomplished by any method by which a nucleic acid molecule canbe inserted into the cell. Transformation techniques include, but arenot limited to, transfection, electroporation, microinjection,lipofection, adsorption, and protoplast fusion. A recombinant cell mayremain unicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid molecules of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of the transformed (i.e., recombinant) cell in sucha manner that their ability to be expressed is retained.

With respect to methods for producing the subject MEKK polypeptide, ahost cell transfected with a nucleic acid vector directing expression ofa nucleotide sequence encoding the subject polypeptides can be culturedunder appropriate conditions to allow expression of the peptide tooccur. The cells may be harvested, lysed and the protein isolated. Acell culture includes host cells, media and other byproducts. Suitablemedia for cell culture are well known in the art. The recombinant MEKKpolypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptide. In a preferred embodiment, therecombinant MEKK polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as GST fusion protein orpoly(His) fusion protein.

Cells of the present invention can be cultured in conventionalfermentation bioreactors, which include, but are not limited to, batch,fed-batch, cell recycle, and continuous fermentors. Culturing can alsobe conducted in shake flasks, test tubes, microtiter dishes, and petriplates. Culturing is carried out at a temperature, pH and oxygen contentappropriate for the recombinant cell. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantMEKK proteins may either remain within the recombinant cell or besecreted into the fermentation medium. The phrase “recovering theprotein” refers simply to collecting the whole fermentation mediumcontaining the protein and need not imply additional steps of separationor purification. MEKK proteins of the present invention can be purifiedusing a variety of standard protein purification techniques, such as,but not limited to, affinity chromatography, ion exchangechromatography, filtration, electrophoresis, hydrophobic interactionchromatography, gel filtration chromatography, reverse phasechromatography, chromatofocusing and differential solubilization.

Alternatively, a MEKK protein of the present invention can be producedby isolating the MEKK protein from cells or tissues recovered from ananimal that normally express the MEKK protein. For example, a cell type,such as T cells, can be isolated from the thymus of an animal. MEKKprotein can then be isolated from the isolated primary T cells usingstandard techniques described herein.

The availability of purified and recombinant MEKK polypeptides asdescribed in the present invention facilitates the development of assayswhich can be used to screen for drugs, including MEKK homologs, whichare either agonists or antagonists of the normal cellular function ofthe subject MEKK polypeptides, or of their role in the pathogenesis ofcellular differentiation and/or proliferation, and disorders relatedthereto. In one embodiment, the assay evaluates the ability of acompound to modulate binding between a MEKK polypeptide and a moleculethat interacts either upstream or downstream of the MEKK polypeptide inthe a cellular signaling pathway. A variety of assay formats willsuffice and, in light of the present inventions, will be comprehended bya skilled artisan.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with upstream ordownstream elements. Accordingly, in an exemplary screening assay of thepresent invention, the compound of interest is contacted with proteinswhich may function upstream (including both activators and repressors ofits activity such as, Ras, Rac, Cdc 42 or Rho or other Ras superfamilymembers) or to proteins or nucleic acids which may function downstreamof the MEKK polypeptide, whether they are positively or negativelyregulated by it. For convenience, such polypeptides of a signaltransduction pathway which interact directly with MEKK will be referredto below as MEKK-binding proteins (MEKK-bp). These proteins include thedownstream targets of MEKKs, namely, members of the MAP kinase kinasefamily (MEKs or MKKs), as MEK1, MEK2, MKK1, MKK2, the stress-activatedkinases (SEKs), also known as the Jun kinase kinases (JNKKs), MEKK3 andMEKK4 or the like. Other downstream targets of the MEKK family caninclude proteins from the mammalian MAP kinase family which includes,for example, the extracellular signal-regulated protein kinases (ERKs),c-Jun NH₂-terminal kinases (JNKs, or SAPKs), and the so-called “p38subgroup” kinases (p38 kinases).

To the mixture of the compound and the MEKK-bp is then added acomposition containing a MEKK polypeptide. Detection and quantificationof complexes including MEKK and the MEKK-bp provide a means fordetermining a compound's efficacy at inhibiting (or potentiating)complex formation between MEKK and the MEKK-binding protein. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide, abaseline for comparison. In the control assay, isolated and purifiedMEKK polypeptide is added to a composition containing the MEKK-bindingprotein, and the formation of a complex is quantitated in the absence ofthe test compound.

In an exemplary embodiment the Ras effector domain or MEKK4 or MEKK4.2sequence IIGQVCDTPKSYDNVMHVGLR is used to inhibit the interaction of aMEKK protein with a MEKK binding protein.

Complex formation between the MEKK polypeptide and a MEKK-bindingprotein may be detected by a variety of techniques. Modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled, fluorescently labeled, orenzymatically labeled MEKK polypeptides, by immunoassay, or bychromatographic detection.

Typically, it will be desirable to immobilize either MEKK or its bindingprotein to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of the two proteins, in the presence and absence of acandidate agent, can be accomplished in any vessel suitable forcontaining the reactants. Examples include microtitre plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows the protein to be boundto a matrix. For example, glutathione-S-transferase/MEKK (GST/MEKK)fusion proteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with the MEKK-bp, e.g. an ³⁵S-labeled, and thetest compound, and the mixture incubated under conditions conducive tocomplex formation, e.g. at physiological conditions for salt and pH,though slightly more stringent conditions may be desired. Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly (e.g. beads placedin scintilant), or in the supernatant after the complexes aresubsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofMEKK-binding protein found in the bead fraction quantitated from the gelusing standard electrophoretic techniques such as described in theappended examples.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either MEKK or itscognate binding protein can be immobilized utilizing conjugation ofbiotin and streptavidin. For instance, biotinylated MEKK molecules canbe prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withMEKK but which do not interfere with binding of upstream or downstreamelements can be derivatized to the wells of the plate, and MEKK trappedin the wells by antibody conjugation. As above, preparations of aMEKK-binding protein and a test compound are incubated in theMEKK-presenting wells of the plate, and the amount of complex trapped inthe well can be quantitated. Exemplary methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the MEKK binding protein, or which are reactive with theMEKK protein and compete with the binding protein; as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the binding protein, either intrinsic or extrinsicactivity. In the instance of the latter, the enzyme can be chemicallyconjugated or provided as a fusion protein with the MEKK-bp. Toillustrate, the MEKK-bp can be chemically cross-linked or geneticallyfused with horseradish peroxidase, and the amount of polypeptide trappedin the complex can be assessed with a chromogenic substrate of theenzyme, e.g. 3,3′-diamino-benzadine terahydrochloride or4-chloro-1-napthol. Likewise, a fusion protein comprising thepolypeptide and glutathione-S-transferase can be provided, and complexformation quantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asanti-MEKK antibodies, can be used. Alternatively, the protein to bedetected in the complex can be “epitope tagged” in the form of a fusionprotein which includes, in addition to the MEKK sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharamacia, N.J.).

In addition to cell-free assays, such as described above, the readilyavailable source of vertebrate MEKK proteins provided by the presentinvention also facilitates the generation of cell-based assays foridentifying small molecule agonists/antagonists and the like. Cellswhich are sensitive to MEKK-mediated signal transduction events can becaused to overexpress a recombinant MEKK protein in the presence andabsence of a test agent of interest, with the assay scoring formodulation in MEKK-dependent responses by the target cell mediated bythe test agent. As with the cell-free assays, agents which produce astatistically significant change in MEKK-dependent signal transduction(either inhibition or potentiation) can be identified.

For example, as described in the appended examples, overexpression ofMEKK1 and MEKK3 (and possibly MEKK2 and MEKK4) in certain cells cancause constitutive induction of apoptotic pathways and result in celldeath. Accordingly, such recombinant cells can be used to identifyinhibitors of MEKK protein signaling by the compound's ability toinhibit signal transduction events downstream of the MEKK proteins andthereby rescue the cell from apoptosis. To illustrate, the recombinantMEKK1 cells of Example 18 or 19 can be contacted with a panel of testcompounds, and inhibitors scored by the ability to rescue the cells froman apoptotic fate (such as may be detected by use of dyes such asHoechst 33258). Compounds which cause a statistically significantdecrease in apoptosis of the MEKK1-overexpressing cells can be selectedfor further testing.

In another embodiment of a drug screening, a two hybrid assay can begenerated with a MEKK and MEKK-binding protein. This assay permits thedetection of protein-protein interactions in yeast such that drugdependent inhibition or potentiation of the interaction can be scored.As an illustrative example, GAL4 protein is a potent activator oftranscription in yeast grown on galactose. The ability of GAL4 toactivate transcription depends on the presence of an N-terminal sequencecapable of binding to a specific DNA sequence (UASG) and a C-terminaldomain containing a transcriptional activator. A sequence encoding aMEKK protein, “A”, may be fused to that encoding the DNA binding domainof the GAL4 protein. A second hybrid protein may be created by fusingsequence encoding the GAL4 transactivation domain to sequence encoding aMEKK-bp, “B”. If protein “A” and protein “B” interact, that interactionserves to bring together the two domains of GALA necessary to activatetranscription of a UASG-containing gene. In addition to co-expressingplasmids encoding both hybrid proteins, yeast strains appropriate forthe detection of protein-protein interactions would contain, forexample, a GAL1-lacZ fusion gene to permit detection of transcriptionfrom a UASG sequence. Other examples of two-hybrid assays or interactiontrap assays are known in the art.

In an illustrative embodiment, a portion of MEKK4 providing a Rac/Cdc42binding site is provided in one fusion protein, along with a secondfusion protein including a Rac/Cdc42 polypeptide. This embodiment of thesubject assay permits the screening of compounds which inhibit orpotentiate the binding of MEKK4 and Cdc42.

Phosphorylation assays may also be used. MEKK binding proteins can betested for their ability to phosphorylate substrates in addition,compounds that inhibit or activate MEKK regulated pathways andphenotypic responses can be tested.

Furthermore, each of the assay systems set out above can be generated ina “differential” format. That is, the assay format can provideinformation regarding specificity as well as potency. For instance,side-by-side comparison of a test compound's effect on different MEKKscan provide information on selectivity, and permit the identification ofcompounds which selectively modulate the bioactivity of only a subset ofthe MEKK family.

The present invention also includes a method to identify compoundscapable of regulating signals initiated from a receptor on the surfaceof a cell, such signal regulation involving in some respect, MEKKprotein. Such a method comprises the steps of: (a) contacting a cellcontaining a MEKK protein with a putative regulatory compound; (b)contacting the cell with a ligand capable of binding to a receptor onthe surface of the cell; and (c) assessing the ability of the putativeregulatory compound to regulate cellular signals by determiningactivation of a member of a MEKK-dependent pathway of the presentinvention. A preferred method to perform step (c) comprises measuringthe phosphorylation of a member of a MEKK-dependent pathway. Suchmeasurements can be performed using immunoassays having antibodiesspecific for phosphotyrosines, phosphoserines and/or phosphothreonines.Another preferred method to perform step (c) comprises measuring theability of the MEKK protein to phosphorylate a substrate moleculecomprising a protein including MKK1, MKK2, MKK3, or MKK4, Raf-1, Ras-GAPand neurofibromin using methods described herein. Preferred substratesinclude MEK1, MEK2, JNKK1 and JNKK2. Yet another preferred method toperform step (c) comprises determining the ability of MEKK protein tobind to Ras, rac or Cdc 42 protein. In particular, determining theability of MEKK protein to bind to GST-Ras^(V12)(GTPγS) orGST-Rac^(v14)(GTPγS).

Putative compounds as referred to herein include, for example, compoundsthat are products of rational drug design, natural products andcompounds having partially defined signal transduction regulatoryproperties. A putative compound can be a protein-based compound, acarbohydrate-based compound, a lipid-based compound, a nucleicacid-based compound, a natural organic compound, a synthetically derivedorganic compound, an anti-idiotypic antibody and/or catalytic antibody,or fragments thereof. A putative regulatory compound can be obtained,for example, from libraries of natural or synthetic compounds, inparticular from chemical or combinatorial libraries (i.e., libraries ofcompounds that differ in sequence or size but that have the samebuilding blocks; see for example, U.S. Pat. Nos. 5,010,175 and 5,266,684of Rutter and Santi) or by rational drug design.

In another embodiment, a method to identify compounds capable ofregulating signal transduction in a cell can comprise the steps of: (a)contacting a putative inhibitory compound with a MEKK protein to form areaction mixture; (b) contacting the reaction mixture with MEK protein;and (c) assessing the ability of the putative inhibitory compound toinhibit phosphorylation of the MEK protein by the MEKK protein. Theresults obtained from step (c) can be compared with the ability of aputative inhibitory compound to inhibit the ability of Raf protein tophosphorylate MEK protein, to determine if the compound can selectivelyregulate signal transduction involving MEKK protein independent of Rafprotein. MEKK, MEK and Raf proteins used in the foregoing methods can berecombinant proteins or naturally-derived proteins.

In another embodiment, a method to identify compounds capable ofregulating signal transduction in a cell can comprise the steps of: (a)contacting a putative inhibitory compound with either a MEKK protein ora Ras superfamily protein, or functional equivalents thereof, to form afirst reaction mixture; (b) combining the first reaction mixture witheither Ras protein (or a functional equivalent thereof) if MEKK proteinwas used in the first reaction mixture, or MEKK protein (or a functionalequivalent thereof) if Raf protein was added to the first reactionmixture; and (c) assessing the ability of the putative inhibitorycompound to inhibit the binding of the Ras protein to the MEKK protein.The lack of binding of the MEKK protein to the Ras protein indicatesthat the putative inhibitory compound is effective at inhibiting bindingbetween MEKK and Ras. MEKK and Ras proteins used in the foregoing methodcan be recombinant proteins or naturally-derived proteins. Preferred Rassuperfamily proteins for use with the foregoing method includes, but isnot limited to, GST-Ras^(V12)(GTPγS) or GST-Rac^(v14)(GTPγS).

The portion of MEKK1, for example, which binds to Ras has beenidentified. The binding of MEKK1 and Ras occurs via the COOH kinasecatalytic domain of MEKK1 and residues 17-42 of Ras as determined by theability of a Ras effector peptide to block the interaction. In addition,the binding of MEKK4.1 and MEKK4.2 to Rac has been localized to theamino acid sequence IIGQVCDTPKSYDNVMHVGLR as described in the appendedExamples. Interestingly this sequence has some homology to the Cdc42/Racinteractive binding (CRIB) region. The consensus CRIB sequence,ISXPXXFXHXXHVG, even with slight variation within this core sequence,confers binding to Cdc42 and/or Rac GTPases (Burbelo et al. (1995) J.Biol Chem 270:29071-29074). Others have postulated that Rac1 is anintermediate between Ha-Ras and MEKK in the signaling cascade leadingfrom growth factor receptors and v-Src to JNK activation based onexperiments with dominant interfering alleles (Minden et al. (1995)Cell. 81:1147-1157).

Preferred MEKK protein for use with the method includes recombinant MEKKprotein. More preferred MEKK protein includes at least a portion of aMEKK protein having the kinase domain of MEKK. Even more preferred MEKKprotein includes a protein encoded by p-MEKK1, MEKK_(COOH), and/orMEKKCOOH-His (see appended Examples). MEKK proteins comprising the aas409-672 of SEQ ID No:2, 1329-1594 of SEQ ID No:4, 361-620 of SEQ ID Nos6 or 8, amino acids 366-626 of SEQ ID No:10, 631-890 of SEQ ID No:12, oramino acids 1338-1597 of SEQ ID No:14 are also preferred.

The inhibition of binding of MEKK protein to Ras superfamily protein canbe determined using a variety of methods known in the art. For example,immunoprecipitation assays can be performed to determine if MEKK and Rasco-precipitate. In addition, immunoblot assays can be performed todetermine if MEKK and Ras co-migrate when resolved by gelelectrophoresis. Another method to determine binding of MEKK to Rascomprises combining a substrate capable of being phosphorylated by MEKKprotein with the Ras protein of the reaction mixture of step (b). Inthis method, Ras protein is separated from the reaction mixture of step(b) following incubation with MEKK protein. If MEKK protein is able tobind to the Ras, then the bound MEKK will be co-isolated with the Rasprotein. The substrate is then added to the isolated Ras protein. Anyco-isolated MEKK protein will phosphorylate the substrate. Thus,inhibition of binding between MEKK and Ras can be measured bydetermining the extent of phosphorylation of the substrate uponcombination with the isolated Ras protein. The extent of phosphorylationcan be determined using a variety of methods known in the art, includingkinase assays using [γ³²P]ATP. Similar assays can be performed with MEKKproteins and their binding to other GTP-binding proteins in the Rassuperfamily (i.e. Rac, Cdc 42, or Rho).

Moreover, one can determine whether the site of inhibitory action alonga particular signal transduction pathway involves both Raf and MEKKproteins by carrying out experiments set forth above (i.e., seediscussion on MEKK-dependent pathways).

Another aspect of the present invention includes a kit to identifycompounds capable of regulating signals initiated from a receptor on thesurface of a cell, such signals involving in some respect, MEKK protein.Such kits include: (a) at least one cell containing MEKK protein; (b) aligand capable of binding to a receptor on the surface of the cell; and(c) a means for assessing the ability of a putative regulatory compoundto alter phosphorylation of the MEKK protein. Such a means for detectingphosphorylation include methods and reagents known to those of skill inthe art, for example, phosphorylation can be detected using antibodiesspecific for phosphorylated amino acid residues, such as tyrosine,serine and threonine. Using such a kit, one is capable of determining,with a fair degree of specificity, the location along a signaltransduction pathway of particular pathway constituents, as well as theidentity of the constituents involved in such pathway, at or near thesite of regulation.

In another embodiment, a kit of the present invention can include: (a)MEKK protein; (b) MEKK substrate, such as MEK; and (c) a means forassessing the ability of a putative inhibitory compound to inhibitphosphorylation of the MEKK substrate by the MEKK protein. A kit of thepresent invention can further comprise Raf protein and a means fordetecting the ability of a putative inhibitory compound to inhibit theability of Raf protein to phosphorylate the MEK protein.

In yet another embodiment, a mammalian MEKK gene can be used to rescue ayeast cell having a defective ste11 (or byr2) gene, such as atemperature sensitive mutant ste11 mutant (cf., Francois et al. (1991) JBiol Chem 266:6174-80; and Jenness et al. (1983) Cell 35:521-9). Forexample, a humanized yeast can be generated by amplifying the codingsequence of the human MEKK clone, and subcloning this sequence into avector which contains a yeast promoter and termination sequencesflanking the MEKK coding sequences. This plasmid can then be used totransform an ste11^(TS) mutant. To assay growth rates, cultures of thetransformed cells can be grown at an permissive temperature for the TSmutant. Turbidity measurements, for example, can be used to easilydetermine the growth rate. At the non-permissive temperature, pheromoneresponsiveness of the yeast cells becomes dependent upon expression ofthe human MEKK protein. Accordingly, the humanized yeast cells can beutilized to identify compounds which inhibit the action of the humanMEKK protein. It is also deemed to be within the scope of this inventionthat the humanized yeast cells of the present assay can be generated soas to comprise other human cell-cycle proteins. For example, human MEKand human MAPK can also be expressed in the yeast cell in place of ste7and Fus3/Kss1. In this manner, the reagent cells of the present assaycan be generated to more closely approximate the natural interactionswhich the mammalian MEKK protein might experience.

Furthermore, certain formats of the subject assays can be used toidentify drugs which inhibit proliferation of yeast cells or other lowereukaryotes, but which have a substantially reduced effect on mammaliancells, thereby improving therapeutic index of the drug as ananti-mycotic agent. For instance, in one embodiment, the identificationof such compounds is made possible by the use of differential screeningassays which detect and compare drug-mediated disruption of bindingbetween two or more different types of MEKK/MEKK-bp complexes, or whichdifferentially inhibit the kinase activity of, for example, ste11relative to a mammalian MEKK. Differential screening assays can be usedto exploit the difference in drug-mediated disruption of human MEKKcomplexes and yeast ste11/byr2 complexes in order to identify agentswhich display a statistically significant increase in specificity fordisrupting the yeast complexes (or kinase activity) relative to thehuman complexes. Thus, lead compounds which act specifically to inhibitproliferation of pathogens, such as fungus involved in mycoticinfections, can be developed. By way of illustration, the present assayscan be used to screen for agents which may ultimately be useful forinhibiting at least one fungus implicated in such mycosis ascandidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis,cryptococcosis, chromoblastomycosis, coccidioidomycosis,conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis,nocaidiosis, para-actinomycosis, penicilliosis, monoliasis, orsporotrichosis. For example, if the mycotic infection to which treatmentis desired is candidiasis, the present assay can comprise comparing therelative effectiveness of a test compound on mediating disruption of ahuman MEKK with its effectiveness towards disrupting the equivalentste11/byr2 kinase from genes cloned from yeast selected from the groupconsisting of Candida albicans, Candida stellatoidea, Candidatropicalis, Candida parapsilosis, Candida krusei, Candidapseudotropicalis, Candida quillermondii, or Candida rugosa. Likewise,the present assay can be used to identify anti-fungal agents which mayhave therapeutic value in the treatment of aspergillosis by making useof genes cloned from yeast such as Aspergillus fumigatus, Aspergillusflavus, Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus.Where the mycotic infection is mucormycosis, the complexes can bederived from yeast such as Rhizopus arrhizus, Rhizopus oryzae, Absidiacorymbifera, Absidia ramosa, or Mucor pusillus. Sources of otherste11/byr2 homologs for comparison with a human MEKK includes thepathogen Pneumocystis carinii.

Another aspect of the present invention relates to the treatment of ananimal having a medical disorder that is subject to regulation or cureby manipulating a signal transduction pathway in a cell involved in thedisorder. Such medical disorders include disorders which result fromabnormal cellular growth or abnormal production of secreted cellularproducts. In particular, such medical disorders include, but are notlimited to, cancer, autoimmune disease, inflammatory responses, allergicresponses and neuronal disorders, such as Parkinson's disease andAlzheimer's disease. Preferred cancers subject to treatment using amethod of the present invention include, but are not limited to, smallcell carcinomas, non-small cell lung carcinomas with overexpressed EGFreceptors, breast cancers with overexpressed EGF or Neu receptors,tumors having overexpressed growth factor receptors of establishedautocrine loops and tumors having overexpressed growth factor receptorsof established paracrine loops. According to the present invention, theterm treatment can refer to the regulation of the progression of amedical disorder or the complete removal of a medical disorder (e.g.,cure). Treatment of a medical disorder can comprise regulating thesignal transduction activity of a cell in such a manner that a cellinvolved in the medical disorder no longer responds to extracellularstimuli (e.g., growth factors or cytokines), or the killing of a cellinvolved in the medical disorder through cellular apoptosis.

According to this aspect of the present invention relates to a method ofinducing and/or maintaining a differentiated state, enhancing survival,and/or promoting (or alternatively inhibiting) proliferation of a cellresponsive to a growth factor, morphogen or other environmental cuewhich effects the cell through at least one signal transduction pathwaywhich includes a MEKK protein. In general, the method comprisescontacting the cells with an amount of an agent which significantly(statistical) modulates MEKK-dependent signaling by the factor. Forinstance, it is contemplated by the invention that, in light of thepresent finding of an apparently broad involvement of members of theMEKK protein family in signal pathways implicated in the formation ofordered spatial arrangements of differentiated tissues in vertebrates,the subject method could be used to generate and/or maintain an array ofdifferent vertebrate tissue both in vitro and in vivo. A “MEKKtherapeutic,” whether inductive or anti-inductive with respect tosignaling by a MEKK-dependent pathway, can be, as appropriate, any ofthe preparations described above, including isolated polypeptides, genetherapy constructs, antisense molecules, peptidomimetics or agentsidentified in the drug assays provided herein.

There are a wide variety of pathological cell proliferative conditionsfor which MEKK therapeutics of the present invention can be used intreatment. For instance, such agents can provide therapeutic benefitswhere the general strategy being the inhibition of an anomalous cellproliferation. Diseases that might benefit from this methodologyinclude, but are not limited to various cancers and leukemias,psoriasis, bone diseases, fibroproliferative disorders such as involvingconnective tissues, atherosclerosis and other smooth muscleproliferative disorders, as well as chronic inflammation.

In addition to proliferative disorders, the present inventioncontemplates the use of MEKK therapeutics for the treatment ofdifferentiative disorders which result from, for example,de-differentiation of tissue which may (optionally) be accompanied byabortive reentry into mitosis, e.g. apoptosis. Such degenerativedisorders include chronic neurodegenerative diseases of the nervoussystem, including Alzheimer's disease, Parkinson's disease, Huntington'schorea, amylotrophic lateral sclerosis and the like, as well asspinocerebellar degenerations. Other differentiative disorders include,for example, disorders associated with connective tissue, such as mayoccur due to de-differentiation of chondrocytes or osteocytes, as wellas vascular disorders which involve de-differentiation of endothelialtissue and smooth muscle cells, gastric ulcers characterized bydegenerative changes in glandular cells, and renal conditions marked byfailure to differentiate, e.g. Wilm's tumors.

It will also be apparent that, by transient use of modulators of MEKKpathways, in vivo reformation of tissue can be accomplished, e.g. in thedevelopment and maintenance of organs. By controlling the proliferativeand differentiative potential for different cells, the subject MEKKtherapeutics can be used to reform injured tissue, or to improvegrafting and morphology of transplanted tissue. For instance, MEKKagonists and antagonists can be employed in a differential manner toregulate different stages of organ repair after physical, chemical orpathological insult. For example, such regimens can be utilized inrepair of cartilage, increasing bone density, liver repair subsequent toa partial hepatectomy, or to promote regeneration of lung tissue in thetreatment of emphysema.

To further illustrate, the present method is applicable to cell culturetechniques. In vitro neuronal culture systems have proved to befundamental and indispensable tools for the study of neural development,as well as the identification of trophic and growth factors such asnerve growth factor (NGF), ciliary trophic factors (CNTF), and brainderived neurotrophic factor (BDNF). Once a neuronal cell has becometerminally-differentiated it typically will not change to anotherterminally differentiated cell-type. However, neuronal cells cannevertheless readily lose their differentiated state. This is commonlyobserved when they are grown in culture from adult tissue, and when theyform a blastema during regeneration. The present method provides a meansfor ensuring an adequately restrictive environment in order to maintainneuronal cells at various stages of differentiation, and can beemployed, for instance, in cell cultures designed to test the specificactivities of other trophic factors. In such embodiments of the subjectmethod, the cultured cells can be contacted with a MEKK therapeutic inorder to induce neuronal differentiation (e.g. of a stem cell), or tomaintain the integrity of a culture of terminally-differentiatedneuronal cells by preventing loss of differentiation. As described inPCT publication PCT/US94/11745, the default fate of ectodermal tissue isneuronal rather than mesodermal and/or epidermal. In particular, it hasbeen reported that preventing or antagonizing signaling by activin canresult in differentiation along a neuronal-fated pathway. The potentialrole of MEKK signaling in mesoderm induction by activin, andconsequently neuronal patterning and development, is further supportedby, for example, LaBonne et al. (1994) Development 120: 463-72, andLaBonne et al. (1995) Development 121: 1475-86. Accordingly, themanipulating the activities of such MAP kinases as the ERKs, p38 kinasesand JNKs, the subject method can be used advantagously to maintain adifferentiated state, or at least to potentiate the activity of amaintenance factor such as CNTF, NGF or the like.

In an exemplary embodiment, the role of the MEKK therapeutic in thepresent method to culture, for example, stem cells, can be to potentiatedifferentiation of uncommitted progenitor cells and thereby give rise toa committed progenitor cell, or to cause further restriction of thedevelopmental fate of a committed progenitor cell towards becoming aterminally-differentiated neuronal cell. For example, the present methodcan be used in vitro as part of a regimen for induction and/ormaintenance of the differentiation of neural crest cells into glialcells, schwann cells, chromaffin cells, cholinergic sympathetic orparasympathetic neurons, as well as peptidergic and serotonergicneurons. The MEKK therapeutic can be used alone, or can be used incombination with other neurotrophic factors which act to moreparticularly enhance a particular differentiation fate of the neuronalprogenitor cell. In the later instance, a MEKK therapeutic might beviewed as ensuring that the treated cell has achieved a particularphenotypic state such that the cell is poised along a certaindevelopmental pathway so as to be properly induced upon contact with asecondary neurotrophic factor. In similar fashion, even relativelyundifferentiated stem cells or primitive neuroblasts can be maintainedin culture and caused to differentiate by treatment with MEKKtherapeutics. Exemplary primitive cell cultures comprise cells harvestedfrom the neural plate or neural tube of an embryo even before much overtdifferentiation has occurred.

Yet another aspect of the present invention concerns the application ofMEKK therapeutics to modulating morphogenic signals involved in othervertebrate organogenic pathways in addition to neuronal differentiation.Thus, it is contemplated by the invention that compositions comprisingMEKK therapeutics can also be utilized for both cell culture andtherapeutic methods involving generation and maintenance of non-neuronaltissue.

In one embodiment, the present invention makes use of the notion thatMEKK proteins are likely to be involved in controlling the developmentand formation of the digestive tract, liver, pancreas, lungs, and otherorgans which derive from the primitive gut. As described in the Examplesbelow, MEKK proteins are presumptively involved in cellular activity inresponse to inductive signals. Additionally, it has been demonstratedthat the activity of a JNK enzyme is markedly stimulated duringregeneration after partial hepatectomy, with a concomitant increase inphosphorylated c-Jun. Accordingly, MEKK agonists and/or antagonists canbe employed in the development and maintenance of an artificial liverwhich can have multiple metabolic functions of a normal liver. In anexemplary embodiment, MEKK therapeutics can be used to induce and/ormaintain differentiation of digestive tube stem cells to form hepatocytecultures which can be used to populate extracellular matrices, or whichcan be encapsulated in biocompatible polymers, to form both implantableand extracorporeal artificial livers.

In another embodiment, compositions of MEKK therapeutics can be utilizedin conjunction with transplantation of such artificial livers, as wellas embryonic liver structures, to promote intraperitoneal implantation,vascularization, and in vivo differentiation and maintenance of theengrafted liver tissue.

Similar utilization of MEKK therapeutics are contemplated in thegeneration and maintenance of pancreatic cultures and artificialpancreatic tissues and organs.

In another embodiment, in vitro cell cultures can be used for theidentification, isolation, and study of genes and gene products that areexpressed in response to disruption of MEKK-mediated signaltransduction, and therefore likely involved in development and/ormaintenance of tissues. These genes would be “downstream” of the MEKKgene products. For example, if new transcription is required for aMEKK-mediated induction, a subtractive cDNA library prepared withcontrol cells and cells overexpressing a MEKK gene can be used toisolate genes that are turned on or turned off by this process. Thepowerful subtractive library methodology incorporating PCR technologydescribed by Wang and Brown is an example of a methodology useful inconjunction with the present invention to isolate such genes (Wang etal. (1991) PNAS 88:11505-11509). Utilizing control and treated cells,the induced pool can be subtracted from the uninduced pool to isolategenes that are turned on, and then the uninduced pool from the inducedpool for genes that are turned off. From this screen, it is expectedthat two classes of mRNAs can be identified. Class I RNAs would includethose RNAs expressed in untreated cells and reduced or eliminated ininduced cells, that is the down-regulated population of RNAs. Class IIRNAs include RNAs that are upregulated in response to induction and thusmore abundant in treated than in untreated cells. RNA extracted fromtreated vs untreated cells can be used as a primary test for theclassification of the clones isolated from the libraries.

In still another embodiment of the present invention, compositionscomprising MEKK therapeutics can be used for the in vitro generation ofskeletal tissue, such as from skeletogenic stem cells, as well as forthe in vivo treatment of skeletal tissue deficiencies. The presentinvention contemplates the use of MEKK therapeutics which upregulate ormimic the inductive activity of a bone morphogenetic protein (BMP) orTGF-β, such as may be useful to control chondrogenesis and/orosteogenesis. By “skeletal tissue deficiency”, it is meant a deficiencyin bone or other skeletal connective tissue at any site where it isdesired to restore the bone or connective tissue, no matter how thedeficiency originated, e.g. whether as a result of surgicalintervention, removal of tumor, ulceration, implant, fracture, or othertraumatic or degenerative conditions, so long as modulation of a TGF-βinductive response is appropriate.

For instance, the present invention makes available effectivetherapeutic methods and MEKK therapeutic compositions for restoringcartilage function to a connective tissue. Such methods are useful in,for example, the repair of defects or lesions in cartilage tissue whichis the result of degenerative wear such as that which results inarthritis, as well as other mechanical derangements which may be causedby trauma to the tissue, such as a displacement of torn meniscus tissue,meniscectomy, a Taxation of a joint by a torn ligament, malignment ofjoints, bone fracture, or by hereditary disease. The present reparativemethod is also useful for remodeling cartilage matrix, such as inplastic or reconstructive surgery, as well as periodontal surgery. Thepresent method may also be applied to improving a previous reparativeprocedure, for example, following surgical repair of a meniscus,ligament, or cartilage. Furthermore, it may prevent the onset orexacerbation of degenerative disease if applied early enough aftertrauma.

The present invention further contemplates the use of the subject methodin the field of cartilage transplantation and prosthetic devicetherapies. To date, the growth of new cartilage from eithertransplantation of autologous or allogenic cartilage has been largelyunsuccessful. Problems arise, for instance, because the characteristicsof cartilage and fibrocartilage varies between different tissue: such asbetween articular, meniscal cartilage, ligaments, and tendons, betweenthe two ends of the same ligament or tendon, and between the superficialand deep parts of the tissue. The zonal arrangement of these tissues mayreflect a gradual change in mechanical properties, and failure occurswhen implanted tissue, which has not differentiated under thoseconditions, lacks the ability to appropriately respond. For instance,when meniscal cartilage is used to repair anterior cruciate ligaments,the tissue undergoes a metaplasia to pure fibrous tissue. By helping tocontrol chondrogenesis, the subject method can be used to particularlyaddresses this problem, by causing the implanted cells to become moreadaptive to the new environment and effectively resemble hypertrophicchondrocytes of an earlier developmental stage of the tissue. Thus, theaction of chondrogensis in the implanted tissue, as provided by thesubject method, and the mechanical forces on the actively remodelingtissue can synergize to produce an improved implant more suitable forthe new function to which it is to be put.

In similar fashion, the subject method can be applied to enhancing boththe generation of prosthetic cartilage devices and to theirimplantation. In one embodiment of the subject method, the implants arecontacted with a MEKK therapeutic during the culturing process so as toinduce and/or maintain differentiated chondrocytes in the culture inorder to further stimulate cartilage matrix production within theimplant. In such a manner, the cultured cells can be caused to maintaina phenotype typical of a chondrogenic cell (i.e. hypertrophic), andhence continue the population of the matrix and production of cartilagetissue.

In another embodiment, the implanted device is treated with a MEKKtherapeutic in order to actively remodel the implanted matrix and tomake it more suitable for its intended function. As set out above withrespect to tissue transplants, the artificial transplants suffer fromthe same deficiency of not being derived in a setting which iscomparable to the actual mechanical environment in which the matrix isimplanted. The activation of the chondrocytes in the matrix by thesubject method can allow the implant to acquire characteristics similarto the tissue for which it is intended to replace.

In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis, as well as inhibits formationof fibrotic tissue proximate the prosthetic device.

In still further embodiments, the subject method can be employed for thegeneration of bone (osteogenesis) at a site in the animal where suchskeletal tissue is deficient. A variety of factors which may signalthrough MEKK proteins are associated with the hypertrophic chondrocytesthat are ultimately replaced by osteoblasts as well as the production ofbone matrix by osteocytes. Consequently, administration of a MEKKtherapeutic can be employed as part of a method for treating bone lossin a subject, e.g. to prevent and/or reverse osteoporosis and otherosteopenic disorders, as well as to regulate bone growth and maturation.For example, preparations comprising MEKK therapeutics can be employed,for example, to induce endochondral ossification by mimicking orpotentiating the activity of a BMP, at least so far as to facilitate theformation of cartilaginous tissue precursors to form the “model” forossification. Therapeutic compositions of such MEKK therapeutics can besupplemented, if required, with other osteoinductive factors, such asbone growth factors (e.g. TGF-β factors, such as the bone morphogeneticfactors BMP-2 and BMP-4, as well as activin), and may also include, orbe administered in combination with, an inhibitor of bone resorptionsuch as estrogen, bisphosphonate, sodium fluoride, calcitonin, ortamoxifen, or related compounds.

In yet another embodiment, treatment with a MEKK therapeutic may permitdisruption of autocrine loops, such as PDGF autostimulatory loops,believed to be involved in the neoplastic transformation of severalneuronal tumors. Modulation of certain of the MEKK proteins may,therefore, be of use to either prevent de-differentiation into mitoticphenotype, or even to induce apoptosis in such cells. Accordingly, thesubject MEKK therapeutics may be useful in the treatment of, forexample, malignant gliomas, medulloblastomas, neuroectodermal tumors,and ependymonas.

For certain cell-types, particularly in epithelial and hemopoieticcells, normal cell proliferation is marked by responsiveness to negativeautocrine or paracrine growth regulators. This is generally accompaniedby differentiation of the cell to a post-mitotic phenotype. However, ithas been observed that a significant percentage of human cancers derivedfrom these cells types display a reduced responsiveness to growthregulators such as TGFβ. For instance, some tumors of colorectal, liverepithelial, and epidermal origin show reduced sensitivity and resistanceto the growth-inhibitory effects of TGFβ as compared to their normalcounterparts. Treatment of such tumors with MEKK therapeutics providesan opportunity to mimic the effective function of TGFβ-mediatedinhibition by constitutive activation of that pathway, and/or offsetother competing pathways which become dominant upon lose of TGFβresponsiveness.

To further illustrate the use of the subject method, the therapeuticapplication of a MEKK therapeutic can be used in the treatment of aneuroglioma. Gliomas account for 40-50% of intracranial tumors at allages of life. Despite the increasing use of radiotherapy, chemotherapy,and sometimes immunotherapy after surgery for malignant glioma, themortality and morbidity rates have not substantially improved. However,there is increasing experimental and clinical evidence that for asignificant number of gliomas, loss of TGFβ responsiveness is animportant event in the loss of growth control. Where the cause ofdecreased responsiveness is due to loss of receptor or loss of otherTGFβ signal transduction downstream of the receptor, treatment with aMEKK therapeutic can be used to constitutively activate the TGFβ pathwayand restore growth inhibition. Alternatively, by manipulation of thelevel activation of the ERKs, apoptosis may be induced.

The subject MEKK therapeutics can also be used in the treatment ofhyperproliferative vascular disorders, e.g. smooth muscle hyperplasia(such as atherosclerosis) or restinosis, as well as other disorderscharacterized by fibrosis, e.g. rheumatoid arthritis, insulin dependentdiabetes mellitus, glomerulonephritis, cirrhosis, and scleroderma,particularly proliferative disorders in which aberrant autocrine orparacrine signaling is implicated.

For example, restinosis continues to limit the efficacy of coronaryangioplasty despite various mechanical and pharmaceutical interventionsthat have been employed. An important mechanism involved in normalcontrol of intimal proliferation of smooth muscle cells appears to bethe induction of autocrine and paracrine TGFβ inhibitory loops in thesmooth muscle cells (Scott-Burden et al. (1994) Tex Heart Inst J21:91-97; Graiger et al. (1993) Cardiovasc Res 27:2238-2247; andGrainger et al. (1993) Biochem J 294:109-112). Loss of sensitivity toTGFβ, or alternatively, the overriding of this inhibitory stimulus suchas by PDGF autostimulation, can be a contributory factor to abnormalsmooth muscle proliferation in restinosis. It may therefore be possibleto treat or prevent restinosis by the use of MEKK therapeutics whichmimic or restore induction by TGFβ or which inhibit PDGF stimulation.

Aberrant signaling by both positive and negative growth regulators alsoplay a significant role in local glomerular and interstitial sites inhuman kidney development and disease. Consequently, the subject methodprovides a method of treating or inhibiting glomerulopathies and otherrenal proliferative disorders comprising the in vivo delivery of asubject MEKK therapeutic.

Yet another aspect of the present invention concerns the therapeuticapplication of a MEKK therapeutic to enhance survival of neurons andother neuronal cells in both the central nervous system and theperipheral nervous system. The ability of signals transduced throughMEKK proteins to regulate neuronal differentiation and survivalindicates that certain of the MEKK proteins can be reasonably expectedto participate in control of adult neurons with regard to maintenance,functional performance, and aging of normal cells; repair andregeneration processes in chemically or mechanically lesioned cells; andprevention of degeneration and premature death which result from loss ofdifferentiation in certain pathological conditions. In light of thisunderstanding, the present invention specifically contemplatesapplications of the subject method to the treatment of (preventionand/or reduction of the severity of) neurological conditions derivingfrom: (i) acute, subacute, or chronic injury to the nervous system,including traumatic injury, chemical injury, vasal injury and deficits(such as the ischemia resulting from stroke), together withinfectious/inflammatory and tumor-induced injury; (ii) aging of thenervous system including Alzheimer's disease; (iii) chronicneurodegenerative diseases of the nervous system, including Parkinson'sdisease, Huntington's chorea, amylotrophic lateral sclerosis and thelike, as well as spinocerebellar degenerations; and (iv) chronicimmunological diseases of the nervous system or affecting the nervoussystem, including multiple sclerosis.

Many neurological disorders are associated with degeneration of discretepopulations of neuronal elements and may be treatable with a therapeuticregimen which includes a MEKK therapeutic. For example, Alzheimer'sdisease is associated with deficits in several neurotransmitter systems,both those that project to the neocortex and those that reside with thecortex. For instance, the nucleus basalis in patients with Alzheimer'sdisease have been observed to have a profound (75%) loss of neuronscompared to age-matched controls. Although Alzheimer's disease is by farthe most common form of dementia, several other disorders can producedementia. Several of these are degenerative diseases characterized bythe death of neurons in various parts of the central nervous system,especially the cerebral cortex. However, some forms of dementia areassociated with degeneration of the thalmus or the white matterunderlying the cerebral cortex. Here, the cognitive dysfunction resultsfrom the isolation of cortical areas by the degeneration of efferentsand afferents. Huntington's disease involves the degeneration ofintrastraital and cortical cholinergic neurons and GABAergic neurons.Pick's disease is a severe neuronal degeneration in the neocortex of thefrontal and anterior temporal lobes, sometimes accompanied by death ofneurons in the striatum. Treatment of patients suffering from suchdegenerative conditions can include the application of MEKKtherapeutics, in order to control, for example, differentiation andapoptotic events which give rise to loss of neurons (e.g. to enhancesurvival of existing neurons) as well as promote differentiation andrepopulation by progenitor cells in the area affected.

In addition to degenerative-induced dementias, a pharmaceuticalpreparation of one or more of the subject MEKK therapeutics can beapplied opportunely in the treatment of neurodegenerative disorderswhich have manifestations of tremors and involuntary movements.Parkinson's disease, for example, primarily affects subcorticalstructures and is characterized by degeneration of the nigrostriatalpathway, raphe nuclei, locus cereleus, and the motor nucleus of vagus.Ballism is typically associated with damage to the subthalmic nucleus,often due to acute vascular accident.

Also included are neurogenic and myopathic diseases which ultimatelyaffect the somatic division of the peripheral nervous system and aremanifest as neuromuscular disorders. In an illustrative embodiment, thesubject method is used to treat amyotrophic lateral sclerosis. ALS is aname given to a complex of disorders that comprise upper and lower motorneurons. Patients may present with progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, or a combination ofthese conditions. The major pathological abnormality is characterized bya selective and progressive degeneration of the lower motor neurons inthe spinal cord and the upper motor neurons in the cerebral cortex. Thetherapeutic application of a MEKK therapeutic, can be used alone, or inconjunction with neurotrophic factors such as CNTF, BDNF or NGF toprevent and/or reverse motor neuron degeneration in ALS patients.

MEKK therapeutics can also be used in the treatment of autonomicdisorders of the peripheral nervous system, which include disordersaffecting the innervation of smooth muscle and endocrine tissue (such asglandular tissue). For instance, the subject method can be used to treattachycardia or atrial cardiac arrythmias which may arise from adegenerative condition of the nerves innervating the striated muscle ofthe heart.

In yet another embodiment, modulation of a MEKK-dependent pathway can beused to inhibit spermatogenesis. Spermatogenesis is a process involvingmitotic replication of a pool of diploid stem cells, followed by meiosisand terminal differentiation of haploid cells into morphologically andfunctionally polarized spermatoza. This process exhibits both temporaland spatial regulation, as well as coordinated interaction between thegerm and somatic cells. It has been previously shown that the signalscoupling extracellular stimulus to regulation of mitotic, meiotic eventswhich occur during spermatogenesis include pathways which rely on, forexample, MAP kinases, for propagation. Accordingly, certain of thesepathways may include MEKK proteins and be alterable by the subject MEKKtherapeutics.

Likewise, members of the MAPK proteins are important in the regulationof female reproductive organs (Wu, T. C. et al. (1994) Mol. Reprod. Dev.38:9-15). Accordingly, certain of the MEKK therapeutics may be useful toprevent oocyte maturation as part of a contraceptive formulation. Inother aspects, regulation of induction of meiotic maturation with MEKKtherapeutics can be used to synchronize oocyte populations for in vitrofertilization. Such a protocol can be used to provide a more homogeneouspopulation of oocytes which are healthier and more viable and more proneto cleavage, fertilization and development to blastocyst stage. Inaddition the MEKK therapeutics could be used to treat other disorders ofthe female reproductive system which lead to infertility includingpolycysitic ovarian syndrome.

Another aspect of the invention features transgenic non-human animalswhich express a heterologous MEKK gene of the present invention, orwhich have had one or more genomic MEKK genes disrupted in at least oneof the tissue or cell-types of the animal. Accordingly, the inventionfeatures an animal model for developmental diseases, which animal hasMEKK allele which is mis-expressed. For example, a mouse can be bredwhich has one or more MEKK alleles deleted or otherwise renderedinactive. Such a mouse model can then be used to study disorders arisingfrom mis-expressed MEKK genes, as well as for evaluating potentialtherapies for similar disorders.

Another aspect of the present invention concerns transgenic animalswhich are comprised of cells (of that animal) which contain a transgeneof the present invention and which preferably (though optionally)express an exogenous MEKK protein in one or more cells in the animal. AMEKK transgene can encode the wild-type form of the protein, or canencode homologs thereof, including both agonists and antagonists, aswell as antisense constructs. In preferred embodiments, the expressionof the transgene is restricted to specific subsets of cells, tissues ordevelopmental stages utilizing, for example, cis-acting sequences thatcontrol expression in the desired pattern. In the present invention,such mosaic expression of a MEKK protein can be essential for many formsof lineage analysis and can additionally provide a means to assess theeffects of, for example, lack of MEKK expression which might grosslyalter development in small patches of tissue within an otherwise normalembryo. Toward this and, tissue-specific regulatory sequences andconditional regulatory sequences can be used to control expression ofthe transgene in certain spatial patterns. Moreover, temporal patternsof expression can be provided by, for example, conditional recombinationsystems or prokaryotic transcriptional regulatory sequences.

Genetic techniques which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that catalyzesthe genetic recombination a target sequence. As used herein, the phrase“target sequence” refers to a nucleotide sequence that is geneticallyrecombined by a recombinase. The target sequence is flanked byrecombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of one of the subject MEKK proteins. For example, excision ofa target sequence which interferes with the expression of a recombinantMEKK gene, such as one which encodes an antagonistic homolog or anantisense transcript, can be designed to activate expression of thatgene. This interference with expression of the protein can result from avariety of mechanisms, such as spatial separation of the MEKK gene fromthe promoter element or an internal stop codon. Moreover, the transgenecan be made wherein the coding sequence of the gene is flanked byrecombinase recognition sequences and is initially transfected intocells in a 3′ to 5′ orientation with respect to the promoter element. Insuch an instance, inversion of the target sequence will reorient thesubject gene by placing the 5′ end of the coding sequence in anorientation with respect to the promoter element which allow forpromoter driven transcriptional activation.

In an illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al.(1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCTpublication WO 92/15694) can be used to generate in vivo site-specificgenetic recombination systems. Cre recombinase catalyzes thesite-specific recombination of an intervening target sequence locatedbetween loxP sequences. loxP sequences are 34 base pair nucleotiderepeat sequences to which the Cre recombinase binds and are required forCre recombinase mediated genetic recombination. The orientation of loxPsequences determines whether the intervening target sequence is excisedor inverted when Cre recombinase is present (Abremski et al. (1984) J.Biol. Chem. 259:1509-1514); catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation expression of a recombinant MEKK protein can be regulatedvia control of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of arecombinant MEKK protein requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and arecombinant MEKK gene can be provided through the construction of“double” transgenic animals. A convenient method for providing suchanimals is to mate two transgenic animals each containing a transgene,e.g., a MEKK gene and recombinase gene.

One advantage derived from initially constructing transgenic animalscontaining a MEKK transgene in a recombinase-mediated expressible formatderives from the likelihood that the subject protein, whether agonisticor antagonistic, can be deleterious upon expression in the transgenicanimal. In such an instance, a founder population, in which the subjecttransgene is silent in all tissues, can be propagated and maintained.Individuals of this founder population can be crossed with animalsexpressing the recombinase in, for example, one or more tissues and/or adesired temporal pattern. Thus, the creation of a founder population inwhich, for example, an antagonistic MEKK transgene is silent will allowthe study of progeny from that founder in which disruption of MEKKmediated induction in a particular tissue or at certain developmentalstages would result in, for example, a lethal phenotype.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the MEKK transgene.Exemplary promoters and the corresponding trans-activating prokaryoticproteins are given in U.S. Pat. No. 4,833,080.

Moreover, expression of the conditional transgenes can be induced bygene therapy-like methods wherein a gene encoding the trans-activatingprotein, e.g. a recombinase or a prokaryotic protein, is delivered tothe tissue and caused to be expressed, such as in a cell-type specificmanner. By this method, a MEKK transgene could remain silent intoadulthood until “turned on” by the introduction of the trans-activator.

In an exemplary embodiment, the “transgenic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonic target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonic target cell. Thezygote is the best target for micro-injection. In the mouse, the malepronucleus reaches the size of approximately 20 micrometers in diameterwhich allows reproducible injection of 1-2 pl of DNA solution. The useof zygotes as a target for gene transfer has a major advantage in thatin most cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster et al. (1985)PNAS 82:4438-4442). Asa consequence, all cells of the transgenic non-human animal will carrythe incorporated transgene. This will in general also be reflected inthe efficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. Microinjection ofzygotes is the preferred method for incorporating transgenes inpracticing the invention.

Retroviral infection can also be used to introduce MEKK transgenes intoa non-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jalmer et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonicstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al.(1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

Methods of making MEKK knock-out or disruption transgenic animals arealso generally known. See, for example, Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).Recombinase dependent knockouts can also be generated, e.g. byhomologous recombination to insert recombinase target sequences flankingportions of an endogenous MEKK gene, such that tissue specific and/ortemporal control of inactivation of a MEKK allele can be controlled asabove.

One aspect of the present invention involves the recognition that a MEKKprotein of the present invention is capable of regulating thehomeostasis of a cell by regulating cellular activity such as cellgrowth cell death, and cell function (e.g., secretion of cellularproducts). Such regulation, in most cases, is independent of Raf,however, as discussed above (and as shown in FIG. 2), some pathwayscapable of regulation by MEKK protein may be subject to upstreamregulation by Raf protein. Therefore, it is within the scope of thepresent invention to either stimulate or inhibit the activity of Rafprotein and/or MEKK protein to achieve desired regulatory results.Without being bound by theory, it is believed that the regulation of Rafprotein and MEKK protein activity at the divergence point from Rasprotein (see FIG. 2) can be controlled by a “2-hit” mechanism. Forexample, a first “hit” can comprise any means of stimulating Rasprotein, thereby stimulating a Ras-dependent pathway, including, forexample, contacting a cell with a growth factor which is capable ofbinding to a cell surface receptor in such a manner that Ras protein isactivated. Following activation of Ras protein, a second “hit” can bedelivered that is capable of increasing the activity of JNK activitycompared with MAPK activity, or vice versa. A second “hit” can include,but is not limited to, regulation of JNK or MAPK activity by compoundscapable of stimulating or inhibiting the activity of MEKK, JNKK (MKK3 orMKK4), Raf and/or MEK. For example, compounds such as protein kinase Cor phospholipase C kinase, can provide the second “hit” needed to drivethe divergent Ras-dependent pathway down the MEKK-dependent pathway insuch a manner that JNK is preferentially activated over MAPK.

One embodiment of the present invention comprises a method forregulating the homeostasis of a cell comprising regulating the activityof a MEKK-dependent pathway relative to the activity of a Raf-dependentpathway in the cell. As used herein, the term “homeostasis” refers tothe tendency of a cell to maintain a normal state using intracellularsystems such as signal transduction pathways. Regulation of the activityof a MEKK-dependent pathway includes increasing the activity of aMEKK-dependent pathway relative to the activity of a Raf-dependentpathway by regulating the activity of a member of a MEKK-dependentpathway, a member of a Raf-dependent pathway, and combinations thereof,to achieve desired regulation of phosphorylation along a given pathway,and thus effect apoptosis. Preferred regulated members of aMEKK-dependent pathway or a Raf-dependent pathway to regulate include,but are not limited to, proteins including MEKK, Ras, Rac, Cdc 42, Raf,MKK, JNKK, MEK, MAPK, JNK, TCF, ATF-2, Jun and Myc, and combinationsthereof.

In one embodiment, the activity of a member of a MEKK-dependent pathway,a member of a Raf-dependent pathway, and combinations thereof, areregulated by altering the concentration of such members in a cell. Onepreferred regulation scheme involves altering the concentration ofproteins including MEKK, Ras, Rac, Cdc 42, Raf, JNKK, MEK, MAPK, JNK,TCF, Jun, ATF-2, and Myc, and combinations thereof. A more preferredregulation scheme involves increasing the concentration of proteinsincluding MEKK, Ras, Rac, Cdc 42, JNKK, JNK, Jun, ATF-2, and Myc, andcombinations thereof. Another more preferred regulation scheme involvesdecreasing the concentration of proteins including Raf, MEK, MAPK, andTCF, and combinations thereof. It is also within the scope of thepresent invention that the regulation of protein concentrations in twoor more of the foregoing regulation schemes can be combined to achievean optimal apoptotic effect in a cell.

A preferred method for increasing the concentration of a protein in aregulation scheme of the present invention includes, but is not limitedto, increasing the copy number of a nucleic acid sequence encoding suchprotein within a cell, improving the efficiency with which the nucleicacid sequence encoding such protein is transcribed within a cell,improving the efficiency with which a transcript is translated into sucha protein, improving the efficiency of post-translational modificationof such protein, contacting cells capable of producing such protein withanti-sense nucleic acid sequences, and combinations thereof.

In a preferred embodiment of the present invention, the homeostasis of acell is controlled by regulating the apoptosis of a cell. A suitablemethod for regulating the apoptosis of a cell is to regulate theactivity of a MEKK-dependent pathway in which the MEKK protein regulatesthe pathway substantially independent of Raf. A particularly preferredmethod for regulating the apoptosis of a cell comprises increasing theconcentration of MEKK protein by contacting a cell with a nucleic acidmolecule encoding a MEKK protein that possesses unregulated kinaseactivity. A preferred nucleic acid molecule with which to contact a cellincludes a nucleic acid molecule encoding a MEKK protein represented bySEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID No:14, and combinations thereof. A more preferred nucleicacid molecule with which to contact a cell includes a nucleic acidmolecule encoding a truncated MEKK protein having only the kinasecatalytic domain (i.e., no regulatory domain) of a MEKK proteinrepresented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12 and/or SEQ ID No:14. An even more preferrednucleic acid molecule with which to contact a cell includes a nucleicacid molecule including the kinase catalytic domain of a MEKK protein,for example, MEKK1.1₄₀₉₋₆₇₂ MEKK1₁₃₂₉₋₁₅₉₄, MEKK2.1₃₆₁₋₆₂₀,MEKK2.2₃₆₁₋₆₂₀ MEKK3₃₆₆₋₆₂₆, MEKK4.1₆₃₁₋₈₉₀, MEKK4.2₁₃₃₈₋₁₅₉₇. Again,suitable variations of MEKK proteins described herein comprise thoseproteins encoded by a nucleic acid molecule that are able to hybridizeto any of the above sequences under stringent conditions.

It is within the scope of the invention that the foregoing method canfurther comprise the step of decreasing the activity of MEK protein inthe cell by contacting the cell with a compound capable of inhibitingMEK activity. Such compounds can include: peptides capable of binding tothe kinase domain of MEK in such a manner that phosphorylation of MAPKprotein by the MEK protein is inhibited; and/or peptides capable ofbinding to a portion of a MAPK protein in such a manner thatphosphorylation of the MAPK protein is inhibited.

In another embodiment, the activity of a member of a MEKK-dependentpathway, a member of a Raf-dependent pathway, and combinations thereof,can be regulated by directly altering the activity of such members in acell. A preferred method for altering the activity of a member of aMEKK-dependent pathway, includes, but is not limited to, contacting acell with a compound capable of directly interacting with a proteinincluding MEKK, Ras, Rac, Cdc 42, JNKK, JNK, Jun, ATF-2, and Myc, andcombinations thereof, in such a manner that the proteins are activated;and/or contacting a cell with a compound capable of directly interactingwith a protein including Raf, MEK, MAPK, TCF protein, and combinationsthereof in such a manner that the activity of the proteins areinhibited. A preferred compound with which to contact a cell that iscapable of regulating a member of a MEKK-dependent pathway includes apeptide capable of binding to the regulatory domain of proteinsincluding MEKK, Ras, Rac, Cdc 42, JNKK, JNK, Jun, ATF-2, and Myc, inwhich the peptide inhibits the ability of the regulatory domain toregulate the activity of the kinase domains of such proteins. Anotherpreferred compound with which to contact a cell includes TNFα, growthfactors regulating tyrosine kinases, hormones regulating Gprotein-coupled receptors and FAS ligand.

A preferred compound with which to contact a cell that is capable ofregulating a member of a Raf-dependent pathway includes a peptidecapable of binding to the kinase catalytic domain of a protein selectedfrom the group consisting of Raf, MEK-1, MEK-2, MAPK, and TCF, in whichthe peptide inhibits the ability of the protein to be phosphorylated orto phosphorylate a substrate.

In accordance with the present invention, a compound can regulate theactivity of a member of a MEKK-dependent pathway by affecting theability of one member of the pathway to bind to another member of thepathway. Inhibition of binding can be achieved by directly interferingat the binding site of either member, or altering the conformationalstructure, thereby precluding the binding between one member and anothermember.

Another preferred compound with which to contact a cell that is capableof regulating a member of a MEKK-dependent pathway includes an isolatedcompound that is capable of regulating the binding of MEKK protein to aprotein of the Ras superfamily, such as Ras, Rac, Cdc 42, or Rho(referred to herein as a Ras:MEKK binding compound). In one embodiment,a Ras:MEKK binding compound of the present invention comprises anisolated peptide (or mimetope thereof) comprising an amino acid sequencederived from a Ras superfamily protein. In another embodiment, aRas:MEKK binding compound of the present invention comprises an isolatedpeptide (or mimetope thereof) comprising an amino acid sequence derivedfrom a MEKK protein.

According to the present invention, an isolated, or biologically pure,peptide, is a peptide that has been removed from its natural milieu. Assuch, “isolated” and “biologically pure” do not necessarily reflect theextent to which the protein has been purified. An isolated compound ofthe present invention can be obtained from a natural source or producedusing recombinant DNA technology or chemical synthesis. As used herein,an isolated peptide can be a full-length protein or any homolog of sucha protein in which amino acids have been deleted (e.g., a truncatedversion of the protein), inserted, inverted, substituted and/orderivatized (e.g., by glycosylation, phosphorylation, acetylation,myristylation, prenylation, palmitilation, and/or amidation) such thatthe peptide is capable of regulating the binding of Ras superfamilyprotein to MEKK protein.

In accordance with the present invention, a “mimetope” refers to anycompound that is able to mimic the ability of an isolated compound ofthe present invention. A mimetope can be a peptide that has beenmodified to decrease its susceptibility to degradation but that stillretain regulatory activity. Other examples of mimetopes include, but arenot limited to, protein-based compounds, carbohydrate-based compounds,lipid-based compounds, nucleic acid-based compounds, natural organiccompounds, synthetically derived organic compounds, anti-idiotypicantibodies and/or catalytic antibodies, or fragments thereof. A mimetopecan be obtained by, for example, screening libraries of natural andsynthetic compounds as disclosed herein that are capable of inhibitingthe binding of Ras superfamily protein to MEKK. A mimetope can also beobtained by, for example, rational drug design. In a rational drugdesign procedure, the three-dimensional structure of a compound of thepresent invention can be analyzed by, for example, nuclear magneticresonance (NMR) or x-ray crystallography. The three-dimensionalstructure can then be used to predict structures of potential mimetopesby, for example, computer modelling. The predicted mimetope structurescan then be produced by, for example, chemical synthesis, recombinantDNA technology, or by isolating a mimetope from a natural source (e.g.,plants, animals, bacteria and fungi).

In one embodiment, a Ras:MEKK binding compound of the present inventioncomprises an isolated peptide having a domain of a Ras superfamilyprotein that is capable of binding to a MEKK protein (i.e., that has anamino acid sequence which enables the peptide to be bound by a MEKKprotein). A Ras peptide of the present invention is of a size thatenables the peptide to be bound by a MEKK protein, preferably, at leastabout 4 amino acid residues, more preferably at least about 12 aminoacid residues, and even more preferably at least about 25 amino acidresidues. In particular, a Ras peptide of the present invention iscapable of being bound by the COOH-terminal region of MEKK, in certainembodiments the region of MEKK containing the MEKK kinase domain.Preferably, a Ras peptide of the present invention comprises theeffector domain of Ras and more preferably amino acid residues 17-42 ofH-Ras. In addition, similar domains of Rac are important for the bindingof Rac, Cdc 42 or Rho to certain MEKK proteins.

In another embodiment, a Ras:MEKK binding compound of the presentinvention comprises an isolated MEKK peptide that has a domain of a MEKKprotein that is capable of binding to a Ras protein (i.e., that has anamino acid sequence which enables the peptide to be bound by a Rasprotein). A MEKK peptide of the present invention is of a size thatenables the peptide to be bound by a Ras protein, in particular by theeffector domain of a Ras protein. Preferably, a MEKK peptide of thepresent invention at least about 320 amino acids in length. Preferably,a MEKK peptide of the present invention comprises the COOH-terminalregion of a MEKK protein and more preferably MEKK_(COOH) (as describedin detail in the appended examples).

As an illustrative example, the sequence of a MEKK protein which bindsto Cdc42 and Rac, such as IIGQVCDTPKSYDNVMHVGLR, occurring aroundresidue 1306-1326 of MEKK4.2 or 599-619 of MEKK4 or mimetics thereofcould be used therapeutically. In one embodiment the Rac-binding portionof a MEKK protein or a fragment thereof is used to block the binding ofthe MEKK catalytic domain with Cdc42 and Rac, thus inhibiting MEKKactivity. Preferred fragment lengths are at least about 4 amino acids,preferably about 8 amino acids, more preferably about 12 amino acids,although longer framents are also contemplated. Similarly the consensusPAK sequence or fragments thereof could be used to block the binding ofMEKK and Cdc42 or Rac. In another embodiment peptidomimetics ormimetopes of these fragments are used. In another embodiment a Raseffector domain peptide is used to blocks the binding of the MEKKcatalytic domain with the GTP-bound form of Ras. Alternatively, theportion of the MEKK catalytic domain which binds to Ras, or the Raseffector domain can be used to competitively inhibit binding of Ras anda MEKK protein.

Ras is a critical component of tyrosine kinase growth factor receptorand G-protein coupled receptor regulation of signal transductionpathways controlling mitogenesis and differentiation. According to thepresent invention, the protein serine-threonine kinases Raf-1 and MEKK1are Ras effectors and selectively bind to Ras in a GTP dependent manner.The p110 catalytic subunit of the lipid kinase has also been shown todirectly interact with Ras in a GTP dependent manner. Ras-GAP andneurofibromin also regulate Ras GTPase activity. Raf-1, MEKK1 andP13-kinase are capable of increasing the activity in cells expressingGTPase-deficient Ras consistent with their interaction with Ras-GTPbeing involved in their regulation.

Different functional domains of Ras effectors bind to Ras in a GTPdependent manner. The Ras binding domain for Raf-1 is encoded in theextreme NH₂-terminal regulatory domain of Raf-1. The Ras binding domainis encoded within the catalytic domain of MEKK1. Both Raf-1 and MEKK1binding to Ras is blocked by a Ras effector domain peptide. Thus, Raf-1,MEKK1 and other Ras effectors can compete for interaction with Ras-GTPpresumably at the Ras effector domain. The relative abundance andaffinity of each Ras effector in different cells may influence themagnitude, onset and duration of each effector response. Secondaryinputs, such as phosphorylation of the different Ras effectors, can alsoinfluence their interaction with Ras-GTP. The kinetic properties of Raseffector activation in cells relative to effector affinity for Ras-GTPare predictable based on the foregoing information. For example, MEKK1can preferentially regulate the SEK/Jun kinase pathways relative toMAPK. Activation of the SEK/Jun kinase pathway is generally slower inonset and maintained as maximal activity longer than the activation ofMAPK.

As additional MEKKs are characterized it will be important tocharacterize their regulation and interaction with other members of theRas superfamily. For example, MEKK4.1 and 4.2 have been found to bind toRac/Cdc42 as described herein. Rho, Rac, and Cdc42 are small GTPasesthat have been implicated in the formation of a variety of actinstructures and the assembly of associated integrin complexes (Burbelo,et al. (1995) J. Biol. Chem. 270:29071-29074). One of the targets of theCdc42 and Rac GTPases is the PAK family of protein kinases (Bagrodia etal (1995) J. Biol. Chem. 270:27995-27998). Rac and Cdc42 have been shownto regulate the activity of the JNK/SAPK signaling pathway in waysdifferent from Ras. While activated Ras stimulates MAPK, but poorlyinduces JNK activity, mutationally activated Rac1 and Cdc42 GTPasespotently activate JNK without affecting MAPK (Coso et al. (1995) Cell81:1137-1146). Undoubtedly additional Ras effectors which interact withand regulate MEKK proteins, perhaps resulting in the selectiveactivation of certain substrates, will be identified in the near future.The present invention also includes a method to administer isolatedcompounds of the present invention to a cell to regulate signaltransduction activity in the cell. In particular, the present inventionincludes a method to administer an isolated compound of the presentinvention to a cell to regulate apoptosis of the cell.

Compounds of the present invention may influence cellular mitogenesis,DNA synthesis, cell division and differentiation. MAPK is alsorecognized as being involved in the activation of oncogenes, such asc-jun and c-myc. While not bound by theory, the present inventorbelieves that MAPK is also intimately involved in various abnormalitieshaving a genetic origin. MAPK is known to cross the nuclear membrane andis believed to be at least partially responsible for regulating theexpression of various genes. As such, MAPK is believed to play asignificant role in the instigation or progression of cancer, neuronaldiseases, autoimmune diseases, allergic reactions, wound healing andinflammatory responses. The present inventor, by being first to identifynucleic acid sequences encoding MEKK, recognized that it is now possibleto regulate the expression of MEKK, and thus regulate the activation ofMAPK.

The present invention also includes a method for regulating thehomeostasis of a cell comprising injecting an area of a subject's bodywith an effective amount of a naked plasmid DNA compound (such as istaught, for example in Wolff et al., 1990, Science 247, 1465-1468). Anaked plasmid DNA compound comprises a nucleic acid molecule encoding aMEKK protein of the present invention, operatively linked to a nakedplasmid DNA vector capable of being taken up by and expressed in arecipient cell located in the body area. A preferred naked plasmid DNAcompound of the present invention comprises a nucleic acid moleculeencoding a truncated MEKK protein having deregulated kinase activity.Preferred naked plasmid DNA vectors of the present invention includethose known in the art. When administered to a subject, a naked plasmidDNA compound of the present invention transforms cells within thesubject and directs the production of at least a portion of a MEKKprotein or RNA nucleic acid molecule that is capable of regulating theapoptosis of the cell.

A naked plasmid DNA compound of the present invention is capable oftreating a subject suffering from a medical disorder including cancer,autoimmune disease, inflammatory responses, allergic responses andneuronal disorders, such as Parkinson's disease and Alzheimer's disease.For example, a naked plasmid DNA compound can be administered as ananti-tumor therapy by injecting an effective amount of the plasmiddirectly into a tumor so that the plasmid is taken up and expressed by atumor cell, thereby killing the tumor cell. As used herein, an effectiveamount of a naked plasmid DNA to administer to a subject comprises anamount needed to regulate or cure a medical disorder the naked plasmidDNA is intended to treat, such mode of administration, number of dosesand frequency of dose capable of being decided upon, in any givensituation, by one of skill in the art without resorting to undueexperimentation.

One aspect of the present invention relates to the recognition that aMEKK protein is capable of activating MAPK and that MAPK can regulatevarious cellular functions as disclosed in U.S. Pat. No. 5,405,941,which is incorporated herein by this reference.

One example of a therapeutic compound of the present invention is thenucleic acid encoding the amino acid residues 1306-1326 of MEKK4.2 or599-619 of MEKK 4. In other embodiments the peptide or fragments thereofcan be used. The Cdc42/Rac binding region of a MEKK peptide(IIGQVCDTPKSYDNVMHVGLR) or the nucleic acid which encodes it can be usedto inhibit the binding of MEKK and a member of the Ras superfamily.Alternatively, the domain of Rac or Cdc42 to which it binds could beused. In another embodiment the region of the Ras effector domain whichblocks the binding of the MEKK catalytic domain with the GTP-bound formof Ras could be used. Alternatively, the portion of the MEKK catalyticdomain which binds to Ras could be used to block MEKK-Ras interaction.

An isolated compound of the present invention can be used to formulate atherapeutic composition. In one embodiment, a therapeutic composition ofthe present invention includes at least one isolated peptide of thepresent invention. A therapeutic composition for use with a treatmentmethod of the present invention can further comprise suitableexcipients. A therapeutic compound for use with a treatment method ofthe present invention can be formulated in an excipient that the subjectto be treated can tolerate. Examples of such excipients include water,saline, Ringer's solution, dextrose solution, Hank's solution, and otheraqueous physiologically balanced salt solutions. Nonaqueous vehicles,such as fixed oils, sesame oil, ethyl oleate, or triglycerides may alsobe used. Other useful excipients include suspensions containingviscosity enhancing agents, such as sodium carboxymethylcellulose,sorbitol, or dextran. Excipients can also contain minor amounts ofadditives, such as substances that enhance isotonicity and chemicalstability. Examples of buffers include phosphate buffer, bicarbonatebuffer and Tris buffer, while examples of preservatives includethimerosal, m- or o-cresol, formalin and benzyl alcohol. Standardformulations can either be liquid injectables or solids which can betaken up in a suitable liquid as a suspension or solution for injection.Thus, in a non-liquid formulation, the excipient can comprise dextrose,human serum albumin, preservatives, etc., to which sterile water orsaline can be added prior to administration.

In another embodiment, a therapeutic compound for use with a treatmentmethod of the present invention can also comprise a carrier. Carriersare typically compounds that increase the half-life of a therapeuticcompound in the treated animal. Suitable carriers include, but are notlimited to, liposomes, micelles, cells, polymeric controlled releaseformulations, biodegradable implants, bacteria, viruses, oils, esters,and glycols. Preferred carriers include liposomes and micelles.

A therapeutic compound for use with a treatment method of the presentinvention can be administered to any subject having a medical disorderas herein described. Acceptable protocols by which to administertherapeutic compounds of the present invention in an effective mannercan vary according to individual dose size, number of doses, frequencyof dose administration, and mode of administration. Determination ofsuch protocols can be accomplished by those skilled in the art withoutresorting to undue experimentation. An effective dose refers to a dosecapable of treating a subject for a medical disorder as describedherein. Effective doses can vary depending upon, for example, thetherapeutic compound used, the medical disorder being treated, and thesize and type of the recipient animal. Effective doses to treat asubject include doses administered over time that are capable ofregulating the activity, including growth, of cells involved in amedical disorder. For example, a first dose of a naked plasmid DNAcompound of the present invention can comprise an amount that causes atumor to decrease in size by about 10% over 7 days when administered toa subject having a tumor. A second dose can comprise at least the samethe same therapeutic compound than the first dose.

Another aspect of the present invention includes a method forprescribing treatment for subjects having a medical disorder asdescribed herein. A preferred method for prescribing treatmentcomprises: (a) measuring the MEKK protein activity in a cell involved inthe medical disorder to determine if the cell is susceptible totreatment using a method of the present invention; and (b) prescribingtreatment comprising regulating the activity of a MEKK-dependent pathwayrelative to the activity of a Raf-dependent pathway in the cell toinduce the apoptosis of the cell. The step of measuring MEKK proteinactivity can comprise: (1) removing a sample of cells from a subject;(2) stimulating the cells with a TNFα; and (3) detecting the state ofphosphorylation of MKK3, MKK4 or JNKK protein using an immunoassay usingantibodies specific for phosphothreonine and/or phosphoserine.

The present invention also includes antibodies capable of selectivelybinding to a MEKK protein of the present invention. Such an antibody isherein referred to as an anti-MEKK antibody. Polyclonal populations ofanti-MEKK antibodies can be contained in a MEKK antiserum. MEKKantiserum can refer to affinity purified polyclonal antibodies, ammoniumsulfate cut antiserum or whole antiserum. As used herein, the term“selectively binds to” refers to the ability of such an antibody topreferentially bind to MEKK proteins. Binding can be measured using avariety of methods known to those skilled in the art includingimmunoblot assays, immunoprecipitation assays, enzyme immunoassays(e.g., ELISA), radioimmunoassays, immunofluorescent antibody assays andimmunoelectron microscopy; see, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989.

Antibodies of the present invention can be either polyclonal ormonoclonal antibodies and can be prepared using techniques standard inthe art. Antibodies of the present invention include functionalequivalents such as antibody fragments and genetically-engineeredantibodies, including single chain antibodies, that are capable ofselectively binding to at least one of the epitopes of the protein usedto obtain the antibodies. Preferably, antibodies are raised in responseto proteins that are encoded, at least in part, by a MEKK nucleic acidmolecule. More preferably antibodies are raised in response to at leasta portion of a MEKK protein, and even more preferably antibodies areraised in response to either the amino terminus or the carboxyl terminusof a MEKK protein. Preferably, an antibody of the present invention hasa single site binding affinity of from about 10³M⁻¹ to about 10¹²M⁻¹ fora MEKK protein of the present invention.

A preferred method to produce antibodies of the present inventionincludes administering to an animal an effective amount of a MEKKprotein to produce the antibody and recovering the antibodies.Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used to identify unique MEKK proteins and recover MEKKproteins.

Another aspect of the present invention comprises a therapeutic compoundcapable of regulating the activity of a MEKK-dependent pathway in a cellidentified by a process, comprising: (a) contacting a cell with aputative regulatory molecule; and (b) determining the ability of theputative regulatory compound to regulate the activity of aMEKK-dependent pathway in the cell by measuring the activation of atleast one member of said MEKK-dependent pathway. Preferred methods tomeasure the activation of a member of a MEKK-dependent pathway includemeasuring the transcription regulation activity of c-Myc protein,measuring the phosphorylation of a protein selected from the groupconsisting of MEKK, JNKK, JNK, Jun, ATF-2, Myc, and combinationsthereof.

Mitogen-activated protein kinase kinase (MEKK1) is a serine/threonineprotein kinase that functions parallel to Raf-1 in the regulation ofsequential protein kinase pathways that involve both mitogen-activatedand stress-activated protein kinases. In this study, we examined theinteraction of MEKK1 with 14-3-3 proteins. The T cell 14-3-3 isoform,but not the β and stratifin isoforms, interacted with MEKK1 in thetwo-hybrid system. GST fusion proteins of the T cell, β, and stratifin14-3-3 isoforms were prepared to further characterize the domains ofMEKK1 and Raf-1 that interact with these proteins. It was demonstratedthat the T cell and β 14-3-3 isoform, but not stratifin, interact withCOS cell-expressed MEKK1. Furthermore, the amino-terminal moiety, butnot the carboxyl-terminal moiety, of expressed MEKK1 interacts with theGST•14-3-3 although the interaction is best when holoMEKK1 is expressed.In contrast, GST•14-3-3 proteins interact with both the amino- andcarboxyl-regions of COS cell-expressed Raf-1 protein. Thus, althoughMEKK1 and Raf-1 function at a parallel point in the sequential proteinkinase pathways, the interaction of 14-3-3 proteins with these kinasesis not identical, suggesting a differential regulation between Raf-1 andMEKK1-stimulated pathways.

The foregoing description of the invention has been presented forpurposes of illustration and description. Further, the description isnot intended to limit the invention to the form disclosed herein.Consequently, variations and modifications commensurate with the aboveteachings, and the skill or knowledge in the relevant art are within thescope of the present invention. The preferred embodiment describedherein above is further intended to explain the best mode known ofpracticing the invention and to enable others skilled in the art toutilize the invention in various embodiments and with variousmodifications required by their particular applications or uses of theinvention. It is intended that the appended claims be construed toinclude alternate embodiments to the extent permitted by the prior art.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1 This Example Describes the StructuralCharacterization of MEKK1 Protein A. MEKK1 Nucleotide Sequence

MEKK1.1 and 1.2 protein was cloned by the following method. Uniquedegenerate inosine oligodeoxynucleotides were designed to correspond toregions of sequence identity between the yeast Ste11 and Byr2 genes.With primers and cDNA templates derived from polyadenylated RNA from NIH3T3 cells, a polymerase chain reaction (PCR) amplification product of320 base pairs (bp) was isolated. This 320 bp cDNA was used as a probeto identify a MEKK1.2 cDNA of 3260 bp from a mouse brain cDNA libraryusing standard methods in the art. The MEKK1.2 nucleotide sequence wasdetermined by dideoxynucleotide sequencing of double-stranded DNA usingstandard methods in the art.

Referring to SEQ ID No:X, based on the Kozak consensus sequence forinitiation codons, the starting methionine can be predicted to occur atnucleotide 486. With this methionine at the start, the cDNA encodes aprotein of 672 amino acids, corresponding to a molecular size of 73 kD.When run on a gel, the protein has an apparent molecular size of 69 kD.There is another in-frame methionine at position 441, which does notfollow the Kozak rule, but would yield a protein of 687 amino acidresidues (74.6 kD). Also referring to SEQ ID No:2, 20% of theNH2-terminal 400 amino acids are serine or threonine and there are onlytwo tyrosines. Several potential sites of phosphorylation by proteinkinase C are apparent in the NH₂-terminal region. The kinase catalyticdomain is located in the COOH-terminal half of the MEKK 1.

B. Southern Blot Analysis of MEKK 1 Transcript

Equal amounts (20 μg) of total RNA were loaded onto the gel as indicatedby ethidium bromide staining. Blots were probed with either a 320-bpcDNA fragment encoding a portion of the MEKK kinase domain or an 858-bpfragment encoding a portion of the NH₂ terminal region of MEKK usingstandard methods in the art. A 7.8 kb mRNA was identified with probesderived from both the 5′ and 3′ ends of the MEKK cDNA in several celllines and mouse tissues. The MEKK mRNA was highly expressed in mouseheart and spleen, an in lower amounts in liver.

C. Southern Blot Analysis

Mouse genomic DNA (10 μg) was digested with either Bam HI, Hind III orEco RI and applied to gels using standard methods in the art. Blots wereprobed with a 320-bp fragment of the MEKK gene. The appearance of oneband was detected in the Bam HI and Hind III digests which indicatesthat MEKK is encoded by one gene. The appearance of two bands in the EcoRI digest indicates the likely presence of an Eco RI site within anintron sequence spanned by the probe.

D. Immunoblots Using Anti-MEKK Antibodies

Three polyclonal antisera were prepared using three different antigens.A first polyclonal antiserum was prepared using an antigen comprising a15 amino acid peptide DRPPSRELLKHPVER derived from the COOH-terminus ofMEKK. NZW rabbits were immunized with the peptide and antisera wasrecovered using standard methods known in the art. This first polyclonalantiserum is hereinafter referred to as the DRPP antiserum.

A second polyclonal antiserum was produced using a DNA clone comprisinga MEKK cDNA digested with EcoR1 and PstI, thereby creating a 1270 bpfragment that encodes the amino terminus of MEKK. This fragment wascloned into pRSETC to form the recombinant molecule pMEKK1-369comprising amino acid residues 1 to 369 of MEKK1. The pMEKK11-369recombinant molecule was expressed in E. coli and protein encoded by therecombinant molecule was recovered and purified using standard methodsknown in the art. NZW rabbits were immunized with the purifiedrecombinant MEKK11-369 protein and antisera was recovered using standardmethods known in the art. This second polyclonal antiserum ishereinafter referred to as the MEKK11-369 antiserum.

A third polyclonal antiserum was produced using a DNA clone comprising aMEKK cDNA digested with Pst I and Kpn 1, thereby creating a 1670 bpfragment that encodes the catalytic domain of MEKK. This fragment wascloned into pRSETC to form the recombinant molecule pMEKK370-738comprising amino acid residues 370 to 738 of MEKK 1 (encoded by basepairs 1592-3260). The pMEKK1370-738 recombinant molecule was expressedin E. coli and protein encoded by the recombinant molecule was recoveredand purified using standard methods known in the art. NZW rabbits wereimmunized with the purified recombinant MEKK1370-738 protein andantisera was recovered using standard methods known in the art. Thissecond polyclonal antiserum is hereinafter referred to as theMEKK1370-738 antiserum.

The DRPP antiserum was used to probe Western Blots of soluble cellularprotein derived from several rodent cell lines. Soluble cellular protein(100 μg) or recombinant MEKK COOH-terminal fusion protein (30 ng) wasloaded onto a 10% Tris Glycine SDS-PAGE gel and the protein transferredto a nylon filter using methods standard in the art. The nylon filterwas immunoblotted with affinity purified DRPP antiserum (1:300dilution). A 78 kD immunoreactive protein was identified in the samplescomprising protein from Pheochromocytoma (PC12), Rat 1a, and NIH 3T3cells. A prominent 50 kD immunoreactive band was also commonly presentbut varied in intensity from preparation to preparation indicating theband is a proteolytic fragment. Visualization of both the 78 kD and 50kD immunoreactive bands on immunoblots was inhibited by pre-incubationof the 15 amino acid peptide antigen with the affinity purified DRPPantiserum. The MEKK protein detected by immunoblotting is similar to themolecular size predicted from the open reading frame of the MEKK cDNA.

In a second immunoblot experiment, PC12 cells stimulated or notstimulated with EGF were lysed and resolved on 10% Tris Glycine SDS-PAGEgel as described above. MEKK proteins contained in the cell lysates wereidentified by immunoblot using affinity purified MEKK11-369 antiserum(1:300) using methods standard in the art. MEKK 1 and two highermolecular weight proteins having MEKK activity, MEKK α and MEKK β, wereidentified using the affinity purified MEKK11-369 antiserum. MEKK 1, andnot MEKK α and MEKK β, were identified using the affinity purifiedMEKK11-369 antiserum.

Using the same procedure described above, two MEKK immunoreactivespecies of approximately 98 kD and 82 kD present in PC12, Rat1a, NIH3T3,and Swiss3T3 cell lysates were recognized by affinity purifiedMEKK11-369 antiserum. It should be noted that the 98 kD MEKK proteindescribed herein was originally identified as a 95 kD MEKK protein inthe related PCT application (International application no.PCT/US94/04178). Subsequent Tris Glycine SDS-PAGE gel analysis has ledto the determination that the modification in molecular weight.Visualization of both of these proteins was inhibited by incubation ofthe affinity purified MEKK11-369 antiserum with purified recombinantMEKK11-369 fusion protein antigen. A single 98 kD MEKK protein waspresent in MEKK immunoprecipitates, but not in immunoprecipitates usingpreimmune serum. More of the 98 kD MEKK was expressed in PC12 cellsrelative to fibroblast cell lines. Immunoblotting with antibodies thatspecifically recognize Raf-1 or Raf-B indicated that neither of theseenzymes were present as contaminants of MEKK immunoprecipitates. 98 kDMEKK in MEKK immunoprecipitates did not comigrate with Raf-1 or Raf-B inPC12 cell lysates and no cross-reactivity between MEKK and Rafantibodies was observed.

Example 2 This Example Describes the Isolation of Nucleic Acid SequencesEncoding MEKK 2, MEKK 3 Proteins and their Activities

Cloning of MEKK 2 and 3. The degenerate primers GA(A/G)(C/T)TIATGGCIGTIAA(A/G)CA (sense) and TTIGCICC(T/C)TTIAT(A/G)TCIC(G/T)(A/G)TG(antisense) were used in a PCR using first strand cDNA generated frompolyadenylated RNA prepared from NIH 3T3 cells. The PCR reactioninvolved 30 cycles (1 min, 94° C.; 2 min, 52° C.; 3 min 72° C.). A bandof approximately 300 base pairs was recovered from the PCR mixture, andthe products were cloned into pGEM-T (Promega). The PCR cDNA productswere sequenced and compared to the MEKK1 sequence. A unique cDNAsequence of 322 base pairs having significant homology to MEKK1 cDNA wasidentified and used to screen an oligo (dT)-primed mouse brain cDNAlibrary (Stratagene). The λ phage library was plated and DNA fromplaques was transferred to Hybond N filters (Amersham) followed byUV-cross-linking of DNA to the filters. Filters were prehybridized for 2h and then hybridized overnight in 0.5M Na₂H₂PO₄ (pH 7.2), 10% bovineserum albumin, 1 mM EDTA, 7% SDS at 68° C. Filters were washed twice at42° C. with 2×SSC, once with 1×SSC, and once with 0.5×SSC containing0.1% SDS. Positive hybridizing clones were purified and sequenced. Toresolve GC-rich regions, cDNAs were subcloned into M13 vectors (NewEngland Biolabs), and single strand DNA was sequenced. In all cases,both strands of DNA were sequenced. MEKK 2 encodes a 619-amino aidprotein having a mass of 69.7 kDa. MEKK 3 encodes a 626-amino acidprotein having a mass of 71 kDa. The two proteins share a commonstructure with the kinase catalytic domain encoded in the COOH-terminalmoiety. The amino-terminal moiety does not encode any definable domainsuch as a SH2 or SH3 domain sequence.

The 5′ ends of both MEKK 2 and 3 are highly G/C-rich making DNAsequencing difficult. To verify the presence of stop codons in all threepossible reading frames 5′ to the predicted start site methionine, theMEKK 2 and 3 cDNAs were inserted in pRSET A, B, and C (Invitrogen) andexpressed in Escherichia coli. Each construct gave a truncated RSETfragment confirming that the MEKK 2 and 3 cDNAs encoded 5′ stop sitesand that the isolated cDNAs encode full-length proteins.

Alignment of the deduced amino acid sequences demonstrated significanthomology between the two proteins. Overall, the two proteins areapproximately 77% homologous. The catalytic domain is encoded in theCOOH-terminal moiety of both MEKK 2 and 3. The first consensus kinasedomain comprising the catalytic site of MEKK 2 and 3 begins at residues361 and 367, respectively. The COOH-terminal catalytic domains of MEKK 2and 3 are approximately 94% conserved, whereas the NH₂-terminal moietiesare only 65% conserved in amino acid sequence. These findings indicatethat the primary sequences of MEKK 2 and 3 diverge significantly in theNH₂-terminal half of the proteins. The conservation in sequence of thecatalytic domains suggests they may recognize an overlapping set ofsubstrates. The divergent NH₂ termini would be consistent with thisregion encoding sequences for the differential regulation of the twoproteins.

The COOH terminus of MEKK 1 encoding the catalytic domain is only 50%homologous to the corresponding regions of MEKK 2 and 3. Thus, thecatalytic domains of MEKK 2 and 3 are very similar to each other butsignificantly divergent from MEKK 1. As shown below, MEKK 1, 2, and 3can all stimulate JNK and p42/44^(MAPK) activities in transfected cells.The significance of the sequence differences in the catalytic domains ofMEKK 1, 2, and 3 is presently unclear.

Plasmid Expression of MEKK2 and 3. The proteins for MEKK2 and 3 wereepitope-tagged at their NH₂ terminus with the hemagglutinin (HA) tagsequence GYPYDVPDYAS using a PCR strategy. For inserting theNH2-terminal epitope tag in MEKK2 and 3, sense oligonucleotides weresynthesized having a methionine codon (ATG), 33 bases coding for theGYPYDVPDYAS epitope tag sequences, and 20 bases of MEKK 2 or 3 sequencestarting at codon 2. For MEKK2, the sense oligonucleotide wasATGGGGTACCCGTACGACGTGCCGGACTACGCTTCCGATGATCAGCAAGCTTTGA A. the senseoligonucleotide for MEKK3 was ATGGGGTACCCGTACGACGTGCCGGACTACGCTTCCGATGAACAAGAGGCATTAGA. The antisense oligonucleotidesfor MEKK2 and 3 were AGACTTAGATCTCAGGTCTTC encoding a BglII site forMEKK2 and GATTCTGACGTCACTCTGCCT encoding an ActII site for MEKK3. ThePCR reactions were performed for 30 cycles using MEKK2 or MEKK3 cDNAs astemplate. The PCR products were purified, and a second PCR reaction wasperformed using the first PCR product as template, the MEKK2 or 3antisense oligonucleotide described above and the common senseoligonucleotide encoding a XbaI restriction site, a consensus Kozakinitiation site and 17 bases overlapping with the initiation methionineand HA tag sequence (TCACGTTCTAGAGCCACCATGGGGTACCCGTACGA). The resultingPCR products were digested with XbaI and BglII for MEKK2 and XbaI andAatII for MEKK3 and ligated in frame into the appropriate MEKK2 or 3cDNA. The sequences were confirmed by DNA sequencing and the cDNAs wereinserted into the expression plasmid pCMV5. HEK 293 cells weretransfected with pCMV5 expression plasmids using Lipofect AMINE (LifeTechnologies, Inc.) and assayed 48 h later. The 12CA5 monoclonalantibody (Berkely Antibody Co.) was used for recognition of the HAepitope tag encoded in expressed MEKK2 and 3.

Antibody Production. Peptides corresponding to COOH-terminal sequencesof MEKK3 (CEARQRPSAEELLTHHFAQ) and p38 (CFVFPPLDQEEMES) were conjugatedto keyhole limpet hemocyanin and used to immunize rabbits. Antisera werecharacterized for specificity by immunoblotting of lysates prepared fromappropriately transfected HEK923 cells.

MEKK 2 and 3 Activate c-Jun Kinase and p42/44^(MAPK) Activity—Transientexpression of MEKK 2 and 3 resulted in the stimulation of c-Jun kinase(JNK) activity. JNK activity was measured using GSTc-Jun₁-79) coupled toglutathione Sepharose 4B. Cells transfected with MEKK2 or 3 and controltransfected cells were lysed in 0.5% Nonidet P-40, 20 mM Tris HCl, pH7.6, 0.25 M NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM dithiothreitol, 1 mM PMSFm2 mM sodium vanadate, 20 ug/ml aprotinin, and 5 ug/ml leupeptin. Nucleiwere removed by centrifugation at 15,000×g for 10 min and thesupernatants (25 ug of protein) were mixed with 10 ul of a slurry of GSTc-Jun(₁₋₇₉) Sepharos (3-5 ug of GST cJun(₁₋₇₉). The mixture was rotatedat 4° C. for 1 h, washed twice in lysis buffer and once in kinase buffer(20 mM Hepes, pH 7.5, 10 mM MgCl₂, 20 mM β-glycerophosphate, 10 mMp-nitrophenyl phosphate, 1 mM dithiothreitol, 50 uM sodium vanadate).Beads were suspended in 40 ul of kinase assay buffer containing 10 uCiof [γ32P]ATP and incubated at 30° C. for 20 min. Reaction mixtures wereadded to Laemmli sample buffer, boiled, and phosphorylated proteins wereresolved on SDS-10% polyacrylamide gels. The JNK activity also elutedearly from a Mono Q column using a linear sodium chloride elutiongradient. Immunoblotting demonstrated that this activity corresponded tothe JNK/stress-activated protein kinase. When JNK activity was assayedfollowing fractionation by Mono Q ion exchange chromatography, 50 ul ofeach fraction was incubated with the GST cJun(₁₋₇₉) beads.

Transient expression of MEKK 2 and 3 also stimulated p42/44^(MAPK)activity. Immunoblotting of hemagglutinin (HA) epitope-tagged MEKK 2 and3 indicated that MEKK 2 and 3 were expressed at similar levels in HEK293cells when 2 pg of plasmid DNA was used per transfection. MAPK activityfollowing Mono Q FPLC fractionation was measured using the epidermalgrowth factor receptor 662-631 peptide as a selective p42/44 MAPKsubstrate. Alternatively, for cells transfected with varying amounts ofMEKK plasmids, MAPK activity was assayed after elution from DEAESephacel columns. To determine whether MEKK 2 and 3 demonstratedselectivity in activating the JNK and p42/44^(MAPK) relative to JNK,plasmid DNAs were titrated over a range of concentrations in thetransfections. MEKK 2 was found to have a greater selectivity forstimulation of the JNK pathway. In contrast, MEKK3 had a greaterselectivity for activating p42/44 MAPK relative to JNK. Thus, eventhough the kinase domains are approximately 94% conserved, MEKK 2 and 3differ in their selectivity for regulation of the JNK and p42/44^(MAPK)pathways. This was particularly evident for MEKK 3 at low plasmidconcentrations where the p42/44^(MAPK) pathway was preferentiallyactivated.

MEKK 2 Phosphorylates Both MEK 1 and JNK Kinase in Vitro. HEK293 cellsexpressing MEKK 2 and 3 were lysed in !% Triton x-100, 0.5% NonidetP-40, 20 mM Tris HCL, pH 7.5, 150 mM NaCl, 20 mM NaF, 0.2 mM sodiumvanadate, 1 mM EDTA, 1 mM EDTA, 1 mM EGTA, 5 mM PMSF. Nuclei wereremoved by centrifugation at 15,000×g for 5 min. HA epitope-tagged MEKK2and 3 were immunoprecipitated with the 12CA5 antibody recognizing the HAepitope-tag. The immunoprecipitates were washed twice in lysis buffer,twice in PAN (10 mM Pipes, pH 7.0, 100 mM NaCl, 20 ug/ml aprotinin),suspended in 20 mM Pipes, 10 mM MnCl₂, 20 ug/ml aprotinin, and used inan in vitro kinase assay with 20-50 ng of recombinant MEK1 or JNKK assubstrates and 20 uCi of [γ³²P]ATP. Reactions were terminated by theaddition of Laemmli sample buffer, boiled, and proteins were resolved bySDS-10% PAGE.

To demonstrate MEKK activation of JNKK activity, the in vitro kinasereactions were performed with different combinations of recombinant,wild type or kinase inactive JNKK (lysine 116 mutated to methionine) andwild type or kinase-inactive JNK. Kinase-inactive JNK was made bymutating the active site lysine 55 to methionine (provided by Dr. MattJarpe). Incubations were for 30 min at 30° C. in the presence of 50 uMATP. GST-cJun (1-79) Sepharose beads were then added, and the mixturewas rotated at 4° C. for 30 min. The beads were washed, suspended in 40ul of c-Jun kinase assay buffer containing 20 uCi of [γ32P]ATP, andincubated for 15 min at 30° C. Reaction mixtures were added to Laemmlisample buffer, boiled, and phosphorylated proteins were resolved on SDS10% PAGE.

MEKK 2 clearly phosphorylates both MEK 1 and JNKK consistent with itsability to activate JNK and p42/44^(MAPK) in HEK298 cells. MEKK2-catalyzed phosphorylation of recombinant JNKK resulted in theenhancement of JNKK activity. Thus, JNKK is a MEKK 2 substrate whoseactivity is stimulated both in vitro and in vivo by MEKK 2. We wereunable to demonstrate the ability of MEKK 3 to phosphorylate MEK 1, MEK2, or JNKK in vitro using a variety of immunoprecipitation procedures.Although MEKK 3 was efficiently immunoprecipitated, as determined byWestern blot analysis, it did not show measurable kinase activity towardMEK 1 or JNKK or show detectable autophosphorylation. This contrasteddramatically with the ability of MEKK 3 to activate both JNK andp42/44^(MAPK) in cells. MEKK 3 protein was clearly immunoprecipitatedusing the 12CA5 antibody in these experiments, and a rabbit antiseraraised against a keyhole limpet hemocyanin-conjugated peptide encodingthe last 15 amino acids of MEKK 3 recognized the intactimmunoprecipitated protein indicating that it was not degraded. Thefailure of immunoprecipitated MEKK 3 to phosphorylate recombinant MEK 1or JNKK suggests one of three possibilities: (i) MEKK 3 is denatured butnot degraded during immunoprecipitation, (ii) MEKK 3 requires anadditional protein or co-factor for its activity in vitro that is lostduring immunoprecipitation, (iii) the relevant substrate for MEKK 3 incells is neither MEK 1 or 2 nor JNKK. At present, it is not clear whichof these possibilities is responsible for the failure to detect MEKK 3activity in vitro. We demonstrated that a mutant MEKK 3 having lysine391 mutated to methionine, rendering it kinase-inactive, did notstimulate JNK or p42/44^(MAPK) activity when expressed in HEK293 cells.This finding indicated that the functional kinase activity of MEKK 3 wasrequired for the in vivo regulation of JNK and p42/44^(MAPK).

MEKK 2 and 3 Do Not Regulate p38 Activity in HEK293 Cells. The p38kinase is activated by hyperosmotic conditions and recognizes thetranscription factor ATF 2 as an in vitro substrate. Sorbitol treated(0.4M, 20 min) or control HEK293 cells were lysed in the same buffer asthat used for immunoprecipitation of p38 using rabbit antiserum raisedagainst the COOH terminal peptide sequence of p38. Immunoprecipitateswere washed once in lysis buffer, once in assay buffer (25 mM Hepes, pH7.4, 25 mM β-glycerophosphate, 25 mM NaCl₂, 2 mM dithiothreitol, 0.1 mMsodium vanadate) resuspended, and used in an in vitro kinase assay witha recombinant NH₂-terminal fragment of ATF 2 (20-50 ng). For analysis ofp38 kinase activity from Mono Q FPLC fractions, 20 ul aliquots weremixed with kinase buffer containing 20-50 ng of recombinant ATF 2 and 10uCi of [γ32P]PATP. Reactions were quenched in Laemmli sample buffer,boiled, and proteins were resolved using SDS 10% PAGE.Immunoprecipitation and in vitro kinase assay of p38 from MEKK 2 and 3transfected HEK293 cells indicated that neither MEKK 2 nor MEKK 3stimulated p38 kinase activity. Mono Q FPLC fractionation of lysatesfrom MEKK 2 or 3 transfected HEK293 cells confirmed that p38 kinaseactivity was similar to that from control transfected cells. ATF 2 isalso a substrate for JNK. Fractions 2-8 from cells transfected with MEKK2 or 3, that contain immunoreactive JNK, have increased kinase activitytoward ATF 2. This is a predicted result based on the ability of bothMEKK 2 and 3 to stimulate JNK activity in HEK293 cells. Expression ofMEKK 2 and 3 also activated additional ATF 2 phosphorylating activitiesresolved by Mono Q fractionation. These activities are seen to elute infractions 9-12 and 13-18 for lysates from both MEKK 2 and 3 expressingcells. These activities do not correspond by immunoblotting to JNK,p42/44^(MAPK), p88, or MEKK 2 or 3 and represent novel kinase activitiescapable of phosphorylating recombinant ATF 2 that are regulated by bothMEKK 2 and 3.

Example 3 This Example Describes the Expression of MEKK 1 Protein inCOS-1 Cells to Define its Function in Regulating the Signaling Systemthat Includes MAPK

COS cells in 100-mm culture dishes were transfected with either thepCVMV5 expression vector alone (1 μg: control) or the pCVMV5 MEKKconstruct (1 μg: MEKK). After 48 hours, the cells were placed inserum-free medium containing bovine serum albumin (0.1 percent) for 16to 18 hours to induce quiescence. Cells were then treated with human EGF(30 ng/ml)(+EGF) or buffer (control) for 10 minutes, washed twice incold phosphate buffered saline (PBS), and lysed in cell lysis buffercontaining 50 mM β-glycerophosphate (pH 7.2), 100 μM sodium vanadate, 2mM MgCl₂, 1 mM EGTA Triton X-100 (0.5 percent), leupeptin (2 μg/ml),aprotinin (2 μg/ml), and 1 mM dithiothreitol (600 μl). Aftercentrifugation for 10 minutes at maximum speed in a microfuge, COS celllysates containing 0.5 to 1 mg of soluble protein were subjected to FPLCon a MONO Q column, and eluted fractions were assayed for MAPK activityaccording to the method described in Heasley et al., p. 545, 1992, Mol.Biol. Cell, Vol. 3.

Referring to FIG. 3, when MEKK 1 was overexpressed in COS1 cells, MAPKactivity was four to five times greater than that in control cellstransfected with plasmid lacking a MEKK 1 cDNA insert. The activation ofMAPK occurred in COS cells deprived of serum and in the absence of anyadded growth factor. The activity of MAPK was similar to that observedafter stimulation of control cells with EGF. Stimulation of COS cellstransiently overexpressing MEKK with EGF resulted in only a slightincrease in MAPK activity compared to that observed with MEKK expressionalone.

To ensure that MEKK protein was present in the samples tested for MAPKactivity, protein from cell lysates of the transfected COS1 cells wereimmunoblotted with MEKK specific antiserum. Equal amounts (100 μg) ofsoluble protein lysate from COS cells were placed on the gel forimmunoblotting using the methods described in Example 1. The filterswere immunoblotted using the affinity purified DRPP antiserum (1:300)and affinity purified MEKK1-369 antiserum (1:300). The results indicatethat expression of MEKK in cells transfected with vector encoding MEKKthat were treated with or without EGF. Only the 50 kD MEKKimmunoreactive fragment was detected in lysates from control COS cellsusing the DRPP antiserum. Transient expression of MEKK in COS cellsyielded a predominant 82 kD band that was slightly larger than thatobserved in PC12, Rat 1a, or NIH 3T3 cells. Addition of the 15 aminoacid DRPP peptide antigen to the antiserum during immunoblottingprevented detection of all of the immunoreactive bands; these bands werenot detected in extracts of control COS cells, an indication that theywere derived from the expressed MEKK protein.

Example 4 This Example Describes the Expression of MEKK1 in Cos Cells toTest the Ability of MEKK Protein to Activate MEK Protein

Recombinant MAPK was used to assay MEK activity in COS cell lysates thathad been fractionated by fast protein liquid chromatography (FPLC) on aMono S column. A cDNA encoding p42 MAPK from Xenopus laevis was clonedinto the pRSETB expression vector. This construct was used forexpression in the LysS strain of Escherichia coli BL21(DE3) of a p42MAPK fusion protein containing a polyhistidine sequence at theNH₂-terminus. Cultures containing the expression plasmid were grown at37° C. to an optical density of 0.7 to 0.9 at 600 nM.Isopropyl-β-thiogalactopyranoside (0.5 mM) was added to induce fusionprotein synthesis and the cultures were incubated for 3 hours. The cellswere then collected and lysed by freezing, thawing, and sonication. Thelysate was centrifuged at 10,000 g for 15 minutes at 4° C. Thesupernatant was then passed over a Ni²⁺⁻ charged Sepharose resin and thesoluble recombinant MAPK was eluted in sodium phosphate buffer (pH 4.5).The purified recombinant MAPK was more than 80 percent pure. Thepurified recombinant MAPK served as a substrate for MEK and catalyzedthe phosphorylation of a peptide consisting of residues 662 to 681 ofthe EGF receptor (EGFR⁶⁶²⁻⁶⁸¹).

Soluble cell lysates from COS cells transiently transfected with MEKK,mock-transfected (control), or mock-transfected and treated with EGF (30ng/ml) (+EGF), were fractionated by FPLC on a Mono S column andendogenous MEK activity was measured. Endogenous MAPK eluted infractions 2 to 4, whereas MEK was contained in fractions 9 to 13. Forassaying endogenous MEK activity, cells were washed twice in cold PBSand lysed in 650 μl of a solution containing 50 mM β-glycerophosphate,10 mM 2-N-morpholinoethane-sulfonic acid (pH 6.0), 100 μM sodiumvanadate, 2 mM MgCl₂, 1 mM EGTA, Triton X-100 (0.5 percent), leupeptin(5 μg/ml), aprotinin (2 μg/ml), and 1 mM dithiothreitol. Aftercentrifugation at maximum speed for 10 minutes in a microfuge, solublecell lysates (1 to 2 mg of protein) were applied to a Mono S columnequilibrated in elution buffer (50 mM β-glycerophosphate, 10 mM MES (pH6.0), 100 μM sodium vanadate, 2 mM MgCl₂, 1 mM EGTA, and 1 mMdithiothreitol. The column was washed with buffer (2 ml) and boundproteins were eluted with a 30 ml linear gradient of 0 to 350 mM NaCl inelution buffer. A portion (30 μl) of each fraction was assayed for MEKactivity by mixing with buffer (25 mM β-glycerophosphate, 40 mMN-(2-hydroxyethyl)piperazine-N′-(2-ethanolsulfonic acid) (pH 7.2) 50 mMsodium vanadate, 10 mM MgCl₂, 100 μM γ-³²P-ATP (3000 to 4000 cpm/pmol),inhibitor protein-20 (IP-20; TTYADFIASGRTGRRNAIHD; 25 μg/ml), 0.5 mMEGTA, recombinant MAP kinase (7.5 μg/ml), and 200 μM EGFR⁶⁶²⁻⁶⁸¹) in afinal volume of 40 μl. After incubation at 30° C. for 20 minutes, theincorporation of γ-³²P-ATP into EGFR⁶⁶²⁻⁶⁸¹ was measured. In this assay,the ability of each column fraction to activate added recombinant MAPKwas measured by the incorporation of γ-³²P-ATP into the MAPK substrate,a peptide derived from the EGF receptor (EGFR).

Referring to FIG. 4, the first peak of activity eluted representsendogenous activated MAPK, which directly phosphorylates the EGFRpeptide substrate. The second peak of activity represents the endogenousMEK in COS cells.

The activity of endogenous MEK activity was characterized byfractionation of Mono S FPLC. COS cell lysates were fractionated by FPLCon a Mono Q column to partially purify the expressed MEKK. Purifiedrecombinant MEK-1 was then used as a substrate for MEKK in the presenceof γ-³²P-ATP to determine whether MEKK directly phosphorylates MEK-1.

A cDNA encoding MEK-1 was obtained from mouse B cell cDNA templates withthe polymerase chain reaction and oligodeoxynucleotide primerscorresponding to portions of the 5′ coding region and 3′ untranslatedregion of MEK-1. The catalytically inactive MEK-1 was generated bysite-directed mutagenesis of Lys343 to Met. The wild-type MEK-1 andcatalytically inactive MEK-1 proteins were expressed in pRSETA asrecombinant fusion proteins containing a polyhistidine sequence at theirNH₂-termini. Lysates from COS cells transfected with MEKK ormock-transfected (control) were subjected to FPLC on a Mono Q column asdescribed above. Portions (20 μl) of fractions containing MEKK weremixed with buffer containing 50 mM β-glycerophosphate (pH 7.2), 100 μMsodium vanadate, 2 mM MgCl₂, 1 mM EGTA, 50 μM ATP, IP-20 (50 μg/ml), and10 μl γ-³²P-ATP in a reaction volume of 40 μl and incubated for 40minutes in the presence (+) or absence (−) of recombinant, catalyticallyinactive MEK-1 (150 ng) (kinase-MEK-1). Reactions were stopped by theaddition of 5×SDS sample buffer (10 μl), 1×SDS buffer contains 2 percentSDS, 5 percent glycerol, 62.5 mM tris-HCl (pH 6.8), 5 percentβ-mercaptoethanol, and 0.001 percent bromophenol blue. The samples wereboiled for 3 minutes and subjected to SDS-PAGE and autoradiography.

Autophosphorylated recombinant wild-type MEK-1 (WT MEK-1) comigratedwith phosphorylated catalytically inactive MEK-1. Thus, MEKK was capableof phosphorylating MEK-1. Corresponding fractions of lysates fromcontrol cells, however, were not able to phosphorylate MEK-1.

Example 5 This Example Describes Studies Showing that the Modified Formof MEK-1 that was Used in the Phosphorylation Assay of Example 4 Did notAutophosphorylate as does Wild-Type MEK-1.

Phosphorylation of catalytically inactive MEK-1 by MEKK was timedependent; MEKK was also phosphorylated. Fraction 22 from FPLC on a MonoQ column (20 μl) was incubated with or without recombinant catalyticallyinactive MEK-1 (0.15 μg) for the indicated times. Phosphorylation ofkinase MEK-1 and MEKK was visable after 5 minutes and maximal afterabout 20 minutes. The time-dependent increase in MEKK phosphorylationcorrelated with a decreased mobility of the MEKK protein duringSDS-PAGE. Immunoblotting demonstrated that the MEKK protein co-eluted(after FPLC on a Mono Q column) with the peak of activity (fraction 22)that phosphorylated MEK. The slowly migrating species of MEKK were alsodetected by immunoblotting. Thus, expression of MEKK appears to activateMAPK by activating MEK.

Example 6 This Example Describes that the Phosphorylation of MEK byOverexpressed MEKK Resulted in Activation of MEK, Recombinant Wild-TypeMEK-1 and a Modified Form of MAPK that is Catalytically Inactive

COS cell lysates were separated by Mono Q-FPLC and fractions containingMEKK were assayed for their ability to activate added wild-type MEK-1such that it would phosphorylate catalytically inactive recombinant MAPKin the presence of γ-³²P-ATP. Lysates from COS cells transfected withMEKK or mock-transfected (control) were fractionated by FPLC on a Mono Qcolumn and portions (20 μl) of fractions containing MEKK were mixed withbuffer. Each fraction was incubated in the presence (+) or absence (−)of purified recombinant wild-type MEK-1 (150 ng) and in the presence ofpurified recombinant, catalytically inactive (kinase-) MAPK (300 ng).Fractions 20 to 24 from lysates of COS cells transfected with MEKKactivated MEK-1. Thus, MEKK phosphorylated and activated MEK-1, leadingto MAPK phosphorylation.

Example 7 This Example Describes Studies Demonstrating that MEKKActivated MEK Directly, and not Through the Activation of One or MoreOther Kinases Contained in the Column Fractions

Overexpressed MEKK was immunoprecipitated from COS cell lysates withaffinity purified MEKK1-369 antiserum. Immunoprecipitated MEKK wasresuspended in 10 to 15 μl of PAN (10 mMpiperazine-N,N′-bis-2-ethanesulfonic acid (Pipes) (pH 7.0), 100 mM NaCl,and aprotinin (20 μg/ml) and incubated with (+) or without (−)catalytically inactive MEK-1 (150 ng) and 25 μCi of γ-³²P-ATP in 20 mMpipes (pH 7.0), 10 mM MnCl₂, and aprotinin (20 μg/ml) in a final volumeof 20 μl for 15 minutes 30° C. Reactions were stopped by the addition of5×SDS sample buffer (5 μl). The samples were boiled for 3 minutes andsubjected to SDS-PAGE and autoradiography.

MEKK phosphorylated catalytically inactive MEK-1, which comigrated withwild-type MEK-1 on SDS-PAGE. Several phosphorylated bands ofoverexpressed MEKK were detected in the immunoprecipitates. These bandsprobably resulted from autophosphorylation of MEKK and corresponded tothe forms of MEKK identified by immunoblotting of lysates from COS cellstransfected with MEKK. Immunoprecipitates obtained with pre-immune serumcontained no MEKK and did not phosphorylate MEK-1. Thus, MEKK appears todirectly phosphorylate MEK.

Taken together, the results from Examples 4 through 7 show that MEKK canphosphorylate and activate MEK, which in turn phosphorylates andactivates MAPK.

Example 8 This Example Demonstrates that Raf can Also Phosphorylate andActivate MEK

COS cells deprived of serum were stimulated with EGF, and Raf wasimmunoprecipitated with an antibody to the COOH-terminus of Raf-1. Coscells were transiently transfected with vector alone (control) or withthe PCV/M5-MEKK construct (MEKK). Quiescent control cells were treatedwith or without human EGF (30 ng/ml) for 10 minutes and Raf wasimmunoprecipitated from cell lysates with an antibody to a COOH-terminalpeptide from Raf. Immunoprecipitated Raf was incubated withcatalytically inactive MEK-1 (150 ng) and 25 μl of γ-³²P-ATP. Theimmunoprecipitated Raf phosphorylated MEK-1 in the presence ofγ-³²P-ATP. Little or no phosphorylation of MEK-1 by Raf was observed inimmunoprecipitates of Raf from COS cells overexpressing MEKK. Treatmentof COS cells overexpressing MEKK with EGF resulted in a similar degreeof phosphorylation of MEK-1 by immunoprecipitated Raf. Cells transfectedwith MEKK and deprived of serum were treated with EGF, and Raf wasimmunoprecipitated and incubated with catalytically inactive MEK-1.Equal amounts of Raf were immunoprecipitated in each sample asdemonstrated by immunoblotting with antibodies to Raf. The slowestmigrating band represents an immunoprecipitated phosphoprotein that isunrelated to Raf or MEK-1. The amount of Raf in the immunoprecipitatesfrom control cells and cells transfected with MEKK was similar as shownby subsequent SDS-PAGE and immunoblotting with the antibody to Raf.Thus, both MEKK and Raf can independently activate MEK.

Example 9 This Example Describes the Activation of a 98 Kd MEKK ProteinIsolated from PC12 Cells in Response to Stimulation of Cells ContainingMEKK Protein by Growth Factors

PC12 cells were deprived of serum by incubation in starvation media(DMEM, 0.1% BSA) for 18-20 hours and MEKK was immunoprecipitated fromlysates containing equal amounts of protein from untreated controls orcells treated with EGF (30 ng/ml) or NGF (10 ng/ml) for 5 minutes withthe above-described anti-MEKK antibodies specific for the NH₄-terminalportion of MEKK. Immunoprecipitated MEKK was resuspended in 8 μl of PAN(10 mM piperazine-N,N′-bis-2-ethanesulfonic acid (Pipes) (pH 7.0), 100mM NaCl, and aprotinin (20 μg/ml)) and incubated with catalyticallyinactive MEK-1 (150 ng) and 40 μCi of (γ-³²P)ATP in universal kinasebuffer (20 mM piperazine-N,N′-bis-2-ethanesulfonic acid (Pipes) (pH7.0), 10 mM MnCl₂, and aprotinin (20 μg/ml)) in a final volume of 20111for 25 minutes at 30° C. Reactions were stopped by the addition of 2×SDSsample buffer (20 μl). The samples were boiled for 3 minutes andsubjected to SDS-PAGE and autoradiography. Raf-B was immunoprecipitatedfrom the same untreated and treated PC12 cell lysates as above with anantiserum to a COOH-terminal peptide of Raf-B (Santa Cruz Biotechnology,Inc.) and assayed similarly. Raf-1 was immunoprecipitated with anantiserum to the 12 COOH-terminal amino acids of Raf-1 (Santa CruzBiotechnology, Inc.). Epidermal growth factor (EGF) treatment of serumstarved PC12 cells resulted in increased MEKK activity.

Referring to FIG. 5, the results were obtained by measuring thephosphorylation of purified MEK-1 (a kinase inactive form) byimmunoprecipitates of MEKK in in vitro kinase assays. NGF stimulated aslight increase in MEKK activity compared to control immunoprecipitatesfrom untreated cells. Stimulation of MEKK activity by NGF and EGF wassimilar to Raf-B activation by these agents, although Raf-B exhibited ahigh basal activity. Activation of c-Raf-1 by NGF and EGF was almostnegligible in comparison to that of MEKK or Raf-B.

A timecourse of MEKK stimulation by EGF was established byimmunoprecipitating MEKK or Raf-B protein from lysates of PC12 cellstreated with EGF (30 ng/ml) for 0, 1, 3, 5, 10, or 20 minutes andincubating the protein with catalytically inactive MEK-1 (150 ng) and(γ-³²P)ATP as described above. Data represent the relative magnitude ofthe response for each timepoint as quantitated by phosphorimageranalysis of radioactive gels from a typical experiment. As shown in FIG.6 a timecourse of EGF treatment indicated that MEKK activation reachedmaximal levels following 5 minutes and persisted for at least 30minutes. Raf-B exhibited a similar timecourse; peak activity occurredwithin 3-5 minutes following EGF treatment and was persistent for up to20 minutes.

To further dissociate EGF-stimulated MEKK activity from that of Raf-B,Raf-B was immunodepleted from cell lysates prior to MEKKimmunoprecipitation. Raf-B was pre-cleared from lysates of serum-starvedPC12 cells which had been either treated or not treated with EGF (30ng/ml) for 5 minutes. Raf-B was pre-cleared two times using antisera toRaf-B or using preimmune IgG antisera as a control. The pre-clearedsupernatant was then immunoprecipitated with either MEKK or Raf-Bantisera and incubated with catalytically inactive MEK-1 and (γ-³²P)ATPas described in detail above. EGF-stimulated and unstimulated PC12 celllysates were precleared with either IgG or Raf-B antisera and thensubjected to immunoprecipitation with MEKK antiserum or Raf-Bantibodies. The results shown in FIG. 7 indicate that pre-clearing withRaf-B resulted in a 60% diminution of Raf-B activity as measured byphosphorimager analysis of Raf-B in vitro kinase assays. EGF-stimulatedMEKK activity was unaffected by Raf-B depletion, suggesting that Raf-Bis not a component of MEKK immunoprecipitates. At least 40% of the Raf-Bactivity is resistant to preclearing with Raf-B antibodies. Recombinantwild type MEKK over-expressed in COS cells readily autophosphorylates onserine and threonine residues and the amino-terminus of MEKK is highlyserine and threonine rich. MEKK contained in immunoprecipitates of PC12cells were tested for selective phosphorylation of purified recombinantMEKK amino-terminal fusion protein in in vitro kinase assays.

Serum-starved PC12 cells were treated with EGF (30 ng/ml) for 5 minutesand equal amounts of protein from the same cell lysates wereimmunoprecipitated with either MEKK, Raf-B, or preimmune antiserum as acontrol. Immunoprecipitates were incubated with purified recombinantMEKK NH₂-terminal fusion protein (400 ng) and (γ-³²P)ATP as describedabove. The results shown in FIG. 8 indicate that MEKK immunoprecipitatedfrom lysates of EGF-stimulated and unstimulated PC12 cells robustlyphosphorylated the inert 50 kD MEKK NH₂-fusion protein, while Raf-B orpreimmune immunoprecipitates from EGF-stimulated or unstimulated cellsdid not use the MEKK NH₂-fusion protein as a substrate. Thus, theEGF-stimulated MEKK activity contained in MEKK immunoprecipites is notdue to contaminating Raf kinases.

Example 10 This Example Describes MEKK Activity in FPLC Mono QIno-Exchange Column Fractions of PC12 Cell Lysates

Cell lysates were prepared from EGF-stimulated PC12 cells. Portions (900μL) of 1 ml column fractions (1 to 525 mM NaCl gradient) wereconcentrated by precipitation with trichloroacetic acid and loaded ongels as described above. The gels were blotted and then immunoblottedwith MEKK specific antibody. The 98 kD MEKK immunoreactivity eluted infractions 10 to 12. The peak of B-Raf immunoreactivity eluted infraction 14, whereas Raf-1 was not detected in the eulates from thecolumn. Portions (30 μl) of each fraction from the PC12 lysates ofunstimulated control cells or EGF-treated cells were assayed asdescribed above in buffer containing purified recombinant MEK-1 (150 ng)as a substrate. These results indicate that the peak of MEKK activityeluted in fractions 10 to 12 from EGF-stimulated PC12 cellsphosphorylated MEK, whereas little MEK phosphorylation occurred infractions from unstimulated cells.

Example 11 This Example Describes Studies Demonstrating that thePhosphorylation of Both MEK-1 and the MEKK NH₂-Terminal Fusion Proteinwere Due to the Activity of the 98 kD PC12 Cell MEKK

Cell lysates prepared from EGF-stimulated and unstimulated cells werefractionated by FPLC on a Mono-Q column to partially purify theendogenous MEKK. Lysates from unstimulated control PC12 cells or cellstreated with EGF (30 ng/ml) for 5 minutes were fractionated by FPLC on aMono Q column using a linear gradient of 0 to 525 mM NaCl. A portion (30μl) of each even numbered fraction was mixed with buffer (20 mMpiperazine-N,N′-bis-2-ethanesulfonic acid (Pipes) (pH 7.0), 10 mM MnCl₂,aprotinin (20 μg/ml), 50 mM β-glycerophosphate (pH 7.2), 1 mM EGTA,IP-20 (50 μg/ml), 50 mM NaF, and 30 μCi (γ-³²P)ATP) containing purifiedrecombinant MEK-1 (150 ng) as a substrate in a final volume of 40 μl andincubated at 30° C. for 25 minutes. Reactions were stopped by theaddition of 2×SDS sample buffer (40 μl), boiled and subjected toSDS-PAGE and autoradiography. The peak of MEKK activity eluted infractions 10-12. Portions (30 μl) of each even numbered fraction fromlysates of EGF-treated PC12 cells were mixed with buffer as describedabove except containing purified recombinant MEKK NH₂-terminal fusionprotein (400 ng) as a substrate instead of MEK-1. Purified recombinantkinase inactive MEK-1 or the MEKK NH₂-terminal fusion protein were thenused as substrates in the presence of (γ-³²P)ATP to determine whether 98kD MEKK directly phosphorylates either substrate. Fractions 10-14 oflysate from PC12 cells treated with EGF phosphorylated MEK-1 whilelittle MEK-1 phosphorylation occurred in untreated control fractions.The MEKK NH₂-terminal fusion protein was also phosphorylated in the samefractions as was MEK-1, although the peak of activity was slightlybroader (fractions 8-16).

Immunoblotting of column fractions demonstrated that the 98 kD MEKKprotein co-eluted with the peak of activity that phosphorylated eitherexogenously added kinase inactive MEK-1 or the 50 kD MEKK NH₂-terminalfusion protein. Portions (900 μl) of even numbered column fractions wereconcentrated by precipitation with trichloroacetic acid andimmunoblotted with MEKK antibody. The peak of immunoreactivity eluted infractions 10-12.

Example 12 This Example Describes the Activation of MEK by a 98 kD MEKK

98 kD MEKK was immunoprecipitated using the MEKK₁₋₃₆₉ antiserumdescribed in Example 1 from untreated (−) or EGF-treated (+) PC12 celllysates. The immunoprecipitates were incubated in the presence (+) orabsence (−) of purified recombinant wild-type MEK (150 ng) and in thepresence of purified recombinant catalytically inactive MAPK (300 ng)and (γ-³²P)ATP. The results indicate that immunoprecipitated MEKK fromEGF-stimulated cells phosphorylated and activated MEK, leading to MAPKphosphorylation. No phosphorylation of MAPK occurred in the absence ofadded recombinant MEK. Immunoblotting demonstrated that there was nocontaminating MAPK or contaminating MEK in the MEKK immunoprecipitatesfrom the EGF-stimulated PC12 cells. Thus, phosphorylation and activationof MEK is due to EGF stimulation of MEKK activity measured in theimmunoprecipitates.

Example 13 This Example Describes Whether 98 kD PC12 Cell MEKK and Raf-BRequire functional Ras proteins for growth factor mediated signaling

Dominant negative Ha-ras (Asn 17) (N¹⁷Ras) was expressed in PC12 cellsand EGF-stimulated MEKK or Raf-B activation was assayed inimmunoprecipitates using kinase inactive MEK-1 as a substrate. PC12cells stably expressing dexamethasone inducible N¹⁷Ras were serumstarved for 18-20 hours in media containing 0.1% BSA with or without 1μM dexamethasone and then untreated or treated with EGF (30 ng/ml) for 5minutes. Equal amounts of soluble protein from cell lysates wasimmunoprecipitated with either MEKK or Raf-B antisera and incubated withpurified recombinant catalytically inactive MEK-1 and (γ-³²P)ATP asdescribed above. Expression of N¹⁷Ras was induced in PC12 clones stablytransfected with the N¹⁷Ras gene by the addition of dexamethasone to thestarvation media. As shown in FIG. 9, N¹⁷Ras expression inhibited theactivation of MEKK by EGF as measured by its ability to phosphorylatekinase inactive MEK. EGF-mediated activation of Raf-B was also greatlyreduced in N¹⁷Ras expressing PC12 cells compared to uninduced N¹⁷Rastransfectants. Addition of dexamethasone to wild type PC12 cells had noeffect on the magnitude of MEKK or Raf-B activation elicited by EGF.PC12 cell clones stably transfected with the N¹⁷Ras gene are lessresponsive to EGF-mediated activation of MEKK activity than are wildtype PC12 cells. These results indicate that functional Ras is requiredfor growth factor stimulated activation of both Raf-B and MEKK in PC12cells, suggesting that Ras may mediate its effects on cell growth anddifferentiation through the activation of multiple protein kinaseeffectors from both the Raf and MEKK families. Thus, EGF stimulated apeak of MEKK activity within 5 minutes which persisted for at least 30minutes following treatment, and was similar to the timecourse of Raf-Bactivation. Nerve growth factor (NGF) and the phorbol ester TPA alsoactivated MEKK, although to a lesser degree than EGF. MEKK activity inimmunoprecipitates or column fractions was dissociable from that ofEGF-stimulated c-Raf-1 and Raf-B activities. Forskolin pretreatmentabolished both MEKK and Raf-B activation by EGF, NGF, and TPA (FIG. 10).Both MEKK and Raf-B activation in response to EGF was inhibited bystable expression of dominant negative N¹⁷ Ras. These findings representthe first demonstration of Ras-dependent MEKK regulation by growthfactors and suggest the emergence of a complex intracellular kinasenetwork in which Ras may alternately couple between members of the Rafand MEKK families.

To determine whether the growth factor-mediated activation of 98 kD PC12cell MEKK was inhibited by PKA, forskolin was used to elevateintracellular cAMP and activate PKA. Serum-starved PC12 cells werepretreated with or without forskolin (50 μM) for 3 minutes to activateprotein kinase A and then with EGF (30 ng/ml), NGF (100 ng/ml), or TPA(200 nM) for 5 minutes and MEKK was immunoprecipitated from equalamounts of soluble protein from cell lysates and incubated with purifiedrecombinant catalytically inactive MEK-1 and (γ-³²P)ATP as describedabove. Raf-B activity was also assayed from the same cell lysates totest whether its regulation differed from that of MEKK. Raf-B wasimmunoprecipitated from the same cell lysates as described above andassayed for its ability to phosphorylate MEK-1 as described above.Forskolin pretreatment abolished the activation of both MEKK and Raf-Bby EGF, NGF, and TPA, as measured by their ability to phosphorylatekinase-inactive MEK-1 (FIG. 10). Forskolin treatment alone had noappreciable effect on either kinase. These results demonstrate that inaddition to Raf-1 and Raf-B, PKA activation inhibits growth factorstimulation of 98 kD PC12 cell MEKK, suggesting the existence of acommon regulatory control point for PKA action which lies between ordownstream of Ras and upstream or at the level of each of these threekinases.

Example 14 This Example Describes the Determination of Whether a Similaror Distinct MEK Activity is Involved in Activation of MAPK Though G_(i)Protein Coupled Receptors by Measuring MEK Activity in Cell Lysates fromThrombin Stimulated Rat 1a Cells

Thrombin stimulated cells exhibited a MEK activity which co-fractionatedwith the major MEK peak detected in EGF stimulated cells. The magnitudeof MEK activity from thrombin challenged cells was generally two tothree-fold less than that observed with EGF stimulation, whichcorrelates with the smaller MAPK response the present inventors haveobserved in thrombin challenged cells.

Differential regulation of MEK in Rat 1a and NIH3T3 cells expressinggip2, v-src, v-ras, or v-raf led the present inventor to investigate theprotein kinases that are putative regulators of MEK-1. Recently, it wasshown that Raf-1 can phosphorylate and activate MEK. Raf activation wasassayed in the following manner. Cells were serum starved and challengedin the presence or absence of the appropriate growth factors, asdescribed above. Serum starved Rat 1a cells were challenged with bufferalone or with EGF and Raf was immunoprecipitated using an antibodyrecognizing the C terminus of Raf. Cells were lysed by scraping in icecold RIPA buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 0.1% SDS, 0.5% sodiumdeoxycholate, 1.0% Triton X 100, 10 mM sodium pyrophosphate, 25 mMsodium glycerophosphate, 2 mM sodium vanadate, 2.1 μg/ml aprotinin) andwere microfuged for 10 min to remove nuclei. The supernatants werenormalized for protein content and precleared with protein A Sepharoseprior to immunoprecipitation with rabbit antiserum to the C terminus ofRaf-1 and protein A Sepharose for 2-3 h at 4° C. The beads were washedtwice with ice cold RIPA and twice with PAN (10 mM Pipes, pH 7.0, 100 mMNaCl, 21 μg/ml aprotinin). A portion of the immunoprecipitate wasdiluted with SDS sample buffer and used for immunoblot analysis. Theremainder was resuspended in kinase buffer (20 mM Pipes pH 7.0, 10 mMMnCl₂, 150 ng kinase-inactive MEK-1, 30 μCi γ-³²P-ATP and 20 μg/mlaprotinin) in a final volume of 50 μl for 30 min at 30° C. Wild typerecombinant MEK-1 was autophosphorylated in parallel as a marker.Reactions were terminated by the addition of 12.5 μl 5×SDS samplebuffer, boiled for 5 minutes and subjected to SDS-PAGE andautoradiography.

The immunoprecipitated Raf, in the presence of γ-³²P-ATP, was able tophosphorylate MEK-1. The recombinant MEK-1 used in this assay was kinaseinactive to ensure it did not autophosphorylate as is observed with wildtype MEK-1. Little or no phosphorylation of MEK-1 by Raf was observed inimmunoprecipitates from control cells. EGF challenge clearly stimulatedRaf catalyzed phosphorylation of MEK-1; in contrast, thrombin challengeof Rat 1a cells did not measurably activate Raf even though endogenousMEK was clearly activated. EGF stimulated Raf phosphorylation ofrecombinant MEK-1 by approximately 2.6-fold over basal. Littlephosphorylation of MEK by Raf was observed in Raf immunoprecipitatesfrom Gip2 or v-Src expressing Rat 1a cells. EGF stimulation was stillcapable of activating Raf catalyzed phosphorylation of MEK-1 in thesecell lines by 1.8 and 1.4-fold, respectively. The blunting of the EFGresponse in Gip2 and v-Src expressing cells is likely a result ofdesensitization of the EFG receptor upon constitutive activation ofMAPK. The amount of Raf in the immunoprecipitates was shown to besimilar by subsequent SDS-PAGE and immunoblotting using Raf antibody.Since thrombin stimulation of MEK is two to three-fold over basal, atleast a 1.5-fold stimulation of MEK phosphorylation is expected if Rafsignificantly contributed to MEK activation by this growth factor. Thislevel of activation was detectable in the EGF stimulated Gip2 and v-Srcexpressing cells lines. Thus, it is unlikely that the failure to detectthrombin activation of Raf is due to the sensitivity of the assay.Thrombin stimulation of MAPK is maximal at 3 minutes. Stimulation of Rat1a cells for 1 or 5 minutes with thrombin did not increase Raf activity.

In NIH3T3 cells, as in Rat 1a cells, EGF activates Raf approximately2.7-fold, while thrombin does not. V-Raf expressing NIH3T3 cells showedno increase in MEK-1 phosphorylation. This result was unexpected sinceMEK was clearly activated in v-Raf expressing NIH3T3 cells. Both the p90and p75 gag-raf fusion proteins in addition to c-Raf-1 wereimmunoprecipitated from v-Raf NIH3T3 cells by the antisera. P75 gag-rafhas been shown to exhibit protein kinase activity, but it is possiblethat the NH₂ terminal gag fusion protein sterically hinders Rafphosphorylation of recombinant MEK-1 in the in vitro assay system.Further studies will have to be done to measure v-Raf kinase activity.The results argue that activation of MEK cannot be accounted forexclusively by the activation of Raf. Additional regulatory kinases forMEK must exist which contribute to MEK activation in thrombinstimulated, G_(i) protein coupled pathways and in gip2 and v-srctransfected cells.

Example 15 This Example Demonstrates the Ability of a PPPSS-Trunc andNco1-Trunc of MEKK Protein to Activate MAPK Activity Compared withFull-Length MEKK Protein and a Negative Control Protein

The results shown in FIG. 11 indicate that the truncated MEKK moleculeswere more active than the full-length MEKK. Indeed, the truncated MEKKmolecules were at least about 1.5 times more active than full-lengthMEKK protein. Thus, removal of the regulatory domain of MEKK deregulatesthe activity of the catalytic domain resulting in improved enzymeactivity.

Example 16 This Example Describes the Preferential Activation of JNK byMEKK Compared with Raf

HeLa cells were transiently transfected with truncated MEKK370-738 undercontrol of an inducible mammary tumor virus promoter, together withepitope tagged JNK1 (described in detail in Derijard et al., p. 1028,1994, Cell, Vol. 76). Other HeLa cells were also transiently transfectedwith truncated BXB-Raf under control of an inducible mammary tumor viruspromoter, together with epitope tagged JNK1 (Derijard et al., ibid.).The following day, MEKK370-738 expression and BXB-Raf expression wereinduced by administration of dexamethasone (10 μM) for 17 hours. Cellextracts were then prepared and assayed for JNK activity using an immunecomplex kinase assay (detailed in Derijard et al., ibid.).Phosphorylation was quantitated by phosphorimager analysis. The resultsshown in FIG. 12 indicate that MEKK stimulated about 30-fold to about50-fold activation more JNK activity over unstimulated cells (basal) andabout 15-fold to about 25-fold JNK activity over Raf stimulated cells.

Example 17 This Example Describes that the Phosphorylation of c-MycTransactivation Domain in Response to MEKK Expression Activates Myc-Gal4 Transcriptional Activity

Two separate expression plasmids were constructed as follows. Theexpression plasmid pLNCX was ligated to a cDNA clone comprising c-myc(1-103) ligated to GAL4 (1-147) (Seth et al., pp. 23521-23524, 1993, J.Biol. Chem., Vol. 266) to form the recombinant molecule pMYC-GAL 4. Theexpression plasmid UAS_(G)-TK Luciferase (Sadowski et al., pp. 563-564,1988, Nature, Vol. 335) was transfected with either pMYC-GAL 4 orpLU-GAL into Swiss 3T3 cells using standard methods in the art to formrecombinant cells herein referred to as LU/GAL cells. Recombinantcontrol cells were also produced by transfecting in pGAL4-Controlplasmids containing GAL4 (1-147) alone in the absence of c-myc (1-103).

LU/Gal cells were transfected with either pMEKK370-738, pMEKK (encodingfull-length MEKK1-738), BXB-Raf, pMyc-Gal4, pCREB-Gal4 (encodingCREB₁₋₂₆₁ fused to Gal 41-147; Hoeffler et al., pp. 868-880, 1989, Mol.Endocrinol., Vol. 3), pGal4, or CREB fusion protein referred to as GAL4.

The transfected cells were incubated overnight and then lysed usingmethods standard in the art. The luciferase activity of each cell lysatewas measure on a luminometer. The results shown in FIG. 13 indicate thatMEKK is selectively capable of stimulating the phosphorylation of c-Myctransactivation domain in such a manner that the c-Myc domain isactivated and induces transcription of the transfected luciferase gene.In addition, the results indicate that MEKK does not stimulate CREBactivation. Also, activated Raf is unable to stimulate Myc activation. Aschematic representation of the activation mechanism of c-Myc protein byMEKK is shown in FIG. 14.

Example 18 This Example Describes the Phosphorylation of P38 MAPKProtein in Response to the Expression of MEKK3 Protein and not MEKK1Protein

COS cells were transfected with the expression plasmid pCVM5 ligated tocDNA clones encoding either MEKK 1 or MEKK 3 protein, or a control pCVM5plasmid lacking MEKK cDNA inserts. Forty-eight hours after transfection,the COS cells were lysed and the lysate fractionated on a Mono Q FPLCcolumn using conditions described in Example 4. The fractions wereanalyzed for tyrosine phosphorylation of MAP kinase-like enzymes usingthe kinase assay described in Example 4. Expression of MEKK 3 inducestyrosine phosphorylation of p38 MAPK and the p42 and p44 forms of MAPK.MEKK 1, however, only induces weak phosphorylation of p38 MAPK but doesinduce phosphorylation of p42 and p44 MAPK.

Example 19 This Example Describes MEKK-Induced Apoptosis

Cells were prepared for the apoptosis studies as follows. Swiss 3T3cells and REF52 cells were transfected with an expression plasmidencoding β-Galactoctosidase (β-Gal) detection of injected cells. One setof β-Gal transfected cells were then microinjected with an expressionvector encoding MEKK370-738 protein. Another set of β-Gal transfectedcells were then microinjected with an expression vector encodingtruncated BXB-Raf protein.

A. Beauvericin-Induced Apoptosis

A first group of transfected Swiss 3T3 cells and REF52 cells weretreated with 50 μM beauvericin for 6 hours at 37° C. Beauvericin is acompound known to induce apoptosis in mammalian cells. A second group ofcells were treated with a control buffer lacking beauvericin. Thetreated cells were then fixed in paraformaldehyde and permeabilized withsaponin using protocols standard in the art. The permeabilized cellswere then labelled by incubating the cells with a fluorescein-labelledanti-tubulin antibody (1:500; obtained from GIBCO, Gaithersburg, Md.) todetect cytoplasmic shrinkage or 10 μM propidium iodide (obtained fromSigma, St. Louis, Mo.) to stain DNA to detect nuclear condensation. Thelabelled cells were then viewed by differential fluorescent imagingusing a Nikon Diaphot fluorescent microscope. The cells treated withbeauvericin demonstrated cytoplasmic shrinkage (monitored by theanti-tubulin antibodies) and nuclear condensation (monitored by thepropidium iodide) characteristic of apoptosis.

B. MEKK-Induced Apoptosis

Swiss 3T3 cells and REF52 cells microinjected with a β-galatoctosidaseexpression plasmid, and an MEKK encoding plasmid or a BXB-Raf encodingplasmid, were treated and viewed using the method described above inSection A. An anti-β-Gal antibody (1:500, obtained from GIBCO,Gaithersburg Md.) was used to detect injected cells. Microscopicanalysis of REF52 cells indicated that the cells expressing MEKK proteinunderwent cytoplasmic shrinkage and nuclear condensation leading toapoptotic death. In contrast, cells expressing BXB-Raf protein displayednormal morphology and did not undergo apoptosis. Similarly, microscopicanalysis of Swiss 3T3 cells indicated that the cells expressing MEKKprotein underwent cytoplasmic shrinkage and nuclear condensation leadingto apoptotic death. In contrast, cells expressing BXB-Raf proteindisplayed normal morphology and did not undergo apoptosis. Thus, MEKKand not Raf protein can induce apoptotic programmed cell death.

Example 20 This Example Describes MEKK-Induced Apoptosis, which isIndependent of JNK/SAPK Activation Methods Microinjection

Swiss 3T3 and REF52 cells were plated on acid-washed glass cover slipsin Dulbecco's Modified Eagle's Medium (DMEM) and 10% bovine calf serum(BCS) or newborn calf serum (NCS). Cells were placed in DMEM/0.1% calfserum for overnight incubation prior to microinjection and used forinjection at 50-70% confluence. Injections were performed with anEppendorf automated microinjection system with needles pulled from glasscapillaries on a vertical pipette puller (Kopf, Tujunga, Calif.). Cellswere injected with pCMVβ-gal in the presence or absence ofpCMV5MEKK_(COOH) or pCMV5BxBRaf at 20-100 ng/μl for each expressionplasmid in 100 mM KC1, 5 mM NaPO₄, pH 7.3. Following injection cellswere placed in 1% NCS for 12-18 hr (Swiss 3T3) or 42 hr (REF52) prior tofixation with paraformaldehyde and staining. Similar results wereobtained when cells were placed in 10% NCS after microinjection.Propidium iodide (5 pg/ml) was used to stain DNA. X-Gal reactions wereperformed for six hr.

Swiss 3T3 cells were microinjected with 100 ng/μl pCMVβ-gal and 20 ng/μlpCMV5MEKK_(COOH). To label free DNA ends fixed and rehydrated cells wereincubated with terminal deoxytransferase (TDT) and 10 nM biotin-dUTPfollowing the manufacturer's instructions (Boehringer-Mannheim). Cellswere stained with FITC-streptavidin to label DNA fragments. β-gal wasdetected using rabbit anti-p-gal antibody (Cappel Labs) and arhodamine-labeled goat anti-rabbit antibody (Cappel Labs).

Transactivation Analysis

Swiss 3T3 cells were transfected using calcium phosphate orlipofectamine with the reporter plasmid Gal4-TK-luciferase, whichcontains four Gal4 binding sites (Sadowski, I., et al. (1988). Nature335, 563-564). adjacent to a minimal thymidine kinase (TK) promoter thatcontrols expression of luciferase, in the presence or absence ofactivator plasmids encoding Gal4₍₁₋₁₄₇₎/Myc₍₇₋₁₀₁₎ (Gupta et al. (1993)Proc. Natl. Acad. Sci. USA 90:3216-3220), Gal4₍₁₋₄₇₎/Elk-1₍₈₃₋₄₂₈₎(Marais, et al. (1993) Cell 73:381-393) or Gal4₍₁₋₁₄₇₎/c-Jun₍₁₋₂₃₃₎ Hibiet al. (1993) Genes & Development 7:2135-2148). Transfections includedpCMV5 without a cDNA insert (basal control), pCMV5MEKK_(COOH) and insome experiments pCMV5BxBRaf. Cells were incubated for 24-48 hr aftertransfection, lysed and assayed for luciferase activity. Values werenormalized to equivalent μg protein for all experiments.

Protein Kinase Assays

JNK/SAPK: Activity was measured using GST (glutathioneS-transferase)—c-Jun (1-79) BOUND to glutathione-Sepharose-4B (Hibi etal. supra). Cells expressing MEKK_(COOH) or control cells were lysed in0.5% Nonidet P40 (NP40), 20 mM Tris-HCl, pH 7.6, 0.25 NaCl, 3 mM EDTA, 3mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 mMsodium vanadate, 20 μg/ml aprotinin and 5 μg/ml leupeptin. Lysates werecentrifuged at 15,000×g for 10 min to remove nuclei and supernatants (25μg protein) mixed with 10 μl of GST-c-JUN₍₁₋₇₉₎-Sepharose (3-5 μg ofGST-c-Jun₍₁₋₇₉₎). The mixture was rotated at 4° C. for 1 hr, washed 2×in lysis buffer and 1× in kinase buffer (20 mM Hepes, pH 7.5, 10 mMMgCl₂, 20 mM β-glycerophosphate, 10 mM p-nitrophenyl phosphate, 1 mMdithiothreitol, 50 μM sodium vanadate). Beads were suspended in 40 μl ofkinase buffer with 10 μCi Of [γ³²P] ATP and incubated at 30° C. for 20min. Samples were boiled in Laemmli buffer and phosphorylated proteinsresolved on SDS/10% polyacrylamide gels. To verify the selectivity ofthe JNK/SAPK assay cell lysates were fractionated by Mono Q ion exchangechromatography and each fraction assayed as described above. Fractionswere also immunoblotted with a rabbit antisera recognizing JNK/SAPK.Only fractions containing immunoreactive JNK/SAPK phosphorylated theGST-c-Jun₍₁₋₇₉₎ protein.p42/44 ERK MAPK: ERK activity was assayed after fractionation of celllysates on DEAE-Sephacel (Heasley, L. E. et al. (1994) Am J. Physiol.267:F366-F373). Alternatively, ERK activity was assayed following Mono Qion exchange chromatography as previously described and characterized(Heasley, et al. (1992) Mol. Biol. Cell. 3:545-553). The EGF receptor662-681 peptide was used as a selective substrate for measuring ERKactivity (Russell, M. et al. (1995) Biochemistry. 34:6611-6615.p38/Hog-1: Cells were lysed in 1% Triton X-100, 0.5% NP40, 20 mMTris-HCl, pH 7.5, 150 mM NaCl, 20 mM NaF, 0.2 mM sodium vanadate, 1 mMEDTA, 1 mM EGTA, 5 mM phenylmethylsulfonyl fluoride. Nuclei were removedby centrifugation at 15,000×g for 5 min. Supernatants (200 μg protein)were used for immunoprecipitation of p38/Hog-1 using rabbit antiserumraised against the COOH-terminal peptide sequence of p38/Hog-1(CFVPPPLDQEEMES) (Han, J. et al. (1992) Mol. Endocrinol. 6:2079-2089)and protein A Sepharose. Immunoprecipitates were washed 1× in lysisbuffer, 1× in assay buffer (25 mM Hepes, pH 7.4, 25 mMP-glycerophosphate, 25 mM NaCl₂, 2 mM dithiothreitol, 0.1 mM sodiumvanadate), resuspended in kinase assay buffer with 20-50 ng of arecombinant NH₂ terminal fragment of ATF-2 as substrate and 20 μCi[γ32P] ATP (Abdel-Hafig, et al. (1992) Mol. Endocrinol. 6:2079-2089).For verification of the immunoprecipitation assay lysates werefractionated by Mono Q ion exchange chromatography and each fractionassayed for ATF-2 kinase activity and immunoblotted with anti-p38antibody. The results demonstrated that p38/Hog-1 containing fractionsselectively phosphorylated the recombinant ATF-2 protein.Competitive Inhibitory Mutant JNK/SAPK and JNKK/SEK-1: The competitiveinhibitory JNK/SAPK mutant referred to JNK/SAPK(APF) had the amino acidsthreonine 183 and tyrosine 185 mutated to alanine and phenylalanine,respectively (Lin et al. (1995) Science 268:286-290). These are thesites phosphorylated by JNKK/SEK-1 and required for activation of theJNK/SAPK kinase activity (Lin et al. supra; Sanchez, I. (1994) Nature372:794-800). Competitive inhibitory JNKK/SEK-1 was made by mutation ofthe active site lysine at residue 116 mutated to an arginine (K116R)rendering the protein kinase inactive (Lin et al. supra).

A. Expression of Activated MEKK Induces Cell Death

Attempts to isolate stable transfectants expressing MEKK_(COOH) inseveral fibroblast lines failed despite repeated attempts. The findingssuggested that expression of activated MEKK inhibited clonal expansionof transfected cells. For this reason, we characterized the functionalconsequence of expressing activated MEKK in Swiss 3T3 and REF52 cellsusing nuclear microinjection of an expression plasmid encoding anactivated form of MEKK1. Cells were microinjected with an expressionplasmid encoding β-galactosidase (β-gal) in the presence or the absenceof the expression plasmid encoding MEKK_(COOH), a truncated activatedform of MEKK1 (Yan, M. et al. (1994) Nature 372:798-800; Lange-Carter,C. A., et al. (1993) Science 260:315-319). When Swiss 3T3 cellsmicroinjected with expression plasmids for β-gal alone (control) orβ-gal plus MEKK_(COOH) it was readily apparent that expression of theactivated MEKK1 induced a strong morphological change of the cells. Incontrast, cells microinjected with the β-gal plasmid alone were similarin morphology to uninjected cells. Injected cells became highlycondensed with a very dark staining of the cytoplasm that hasdramatically shrunken relative to the flattened morphology of the cellsinjected with β-gal alone. The results indicated MEKK_(COOH) expressionresulted in death of the cells.

For further analysis and comparison cells were microinjected withBxBRaf, a truncated activated form of Raf-1 (Rapp, U. R. (1991) Oncogene6:495-500) that selectively activates the ERK pathway (Kyriakis, J. M.et al. (1992) Nature 358:417-421). In microinjected cells, expression ofβ-gal, MEKK_(COOH) or BxBRaf was demonstrated by indirectimmunofluorescence using specific antibodies recognizing each protein.Swiss 3T3 cells and REF 52 cells microinjected with the indicatedexpression plasmid were fixed and stained only eight hours postinjectionto demonstrate that each protein was being expressed in the cytoplasm ofthe cells. It was apparent with the REF 52 cells expressing MEKK beganto undergo a morphological changes relative to β-gal expressing cells.

TABLE 1 Quantitation of MEKK_(COOH)-induced cell death. DNA InjectedCells Injected Condensed Cells β-gal 336 4 (1%) β-gal+ 175 5 (3%) BxBRafβ-gal+ 200 167 (84%) MEKK_(COOH) β-gal+ 50 0 (0%) Kin~MEKK_(COOH)Swiss 3T3 cells were injected with solutions containing 100 ng/μlCMV-βgal in the presence or absence of 100 ng/μl of pCMV5-BxEBRaf,pCMV5-MEKK_(COOH) or pCMV5-Kin˜MEKK_(COOH) (kinase inactive mutant; 13).Seventeen hours after injection cells were fixed and stained for,β-galactosidase activity with X-Gal. Injected cells attached to thecoverslip were scored as positive for cell death when they were highlycondensed, small round cells.

The results of this experiment demonstrated that expression ofMEKK_(COOH) resulted in significant cell death characterized by thedramatic morphological condensation. In contrast, BxBRaf expression didnot affect cell viability relative to control cells expressing onlyβ-gal. Approximately 84% of all MEKK_(COOH) injected cells had a highlycondensed cellular morphology seventeen hours after injection. Thiscount actually underestimates the number of condensed cells becauseSwiss 3T3 cells in advanced stages of the cell death response were oftennonadherent to coverslips. Some of the nonadherent highly condensedcells could be found to be released from the coverslip into the culturemedium, but were not scored in the quantitation. In contrast, fewer than3% of BxBRaf and 1% of control β-gal injected cells had an alteredmorphology even after 48-72 hours post-injection.

These data also show that cell death resulting from MEKK_(COOH)expression required the kinase activity of the enzyme; the kinaseinactive mutant of MEKK_(COOH) was without effect. The apoptotic-likecell death was also dependent on the MEKK_(COOH) concentration asmeasured by serial dilution (0-100 ng/μl) of the expression plasmid usedfor microinjection. Maintenance of the MEKK_(COOH) expressing cells in10% serum slightly prolonged the time required for induction ofcytoplasmic shrinkage, nuclear condensation and cell death suggestingthat growth factors and cytokines had some influence on the onset of theresponse induced by MEKK_(COOH) but high serum could not preventMEKK_(COOH) induced cell death. Greater than 80% of MEKK_(COOH)expressing cells had a cytoplasmic and nuclear morphology characteristicof apoptosis 18 hrs postinjection.

More dramatic morphological changes in Swiss 3T3 cells also resultedfrom expression of MEKK_(COOH). Cytoplasmic shrinkage is evident fromthe β-gal staining and nuclear condensation is evident in MEKK1expressing cells stained with propidium iodide. In contrast, cellsexpressing BxBRaf do not demonstrate any detectable morphologicaldifference from control cells expressing only β-gal. Similar dramaticcytoplasmic shrinkage and nuclear condensation was observed withMEKK_(COOH) expression in REF52 cells, where BxBRaf again had no effecton cytoplasmic and nuclear integrity. To assess if DNA fragmentation wasinduced by MEKK_(COOH) expression, terminal deoxytransferase (TDT) wasused to covalently transfer biotin-dUTP to the ends of DNA breaks insitu. Streptavidin-FITC was then used for detection of dUTP incorporatedinto cellular DNA. Even though Swiss 3T3 cells do not undergosignificant DNA degradation and laddering at the nucleosomal level theydo generate larger DNA fragments when stimulated to undergo apoptosis(Obeid, L. M. et al. (1993). Science 259:1769-1771). The condensednuclei of MEKK_(COOH) injected cells were highly fluorescent indicatingsignificant DNA fragmentation. It is also apparent that the cytoplasmhas become highly condensed and the condensed chromatin is distinct fromthe cytoplasm. Microinjected cells not yet undergoing cytoplasmic andnuclear condensation in response to MEKK_(COOH) did not incorporate dUTPinto their DNA. Thus, expression of MEKK_(COOH) induced all thehallmarks of apoptosis including cytoplasmic shrinkage, nuclearcondensation and DNA fragmentation.

Expression of BxBRaf did not induce a response measured by any of thecriteria mentioned above. BxBRaf expressing cells displayed a normalflattened morphology similar to β-gal expressing cells or to uninjectedcells. Transient BxBRaf expression in Swiss 3T3 cells stimulated ERKactivity (not shown) and the transactivation function of the Gal4/Elk-1chimeric transcription factor, shown in FIG. 15, whose activation isdependent on phoshorylation by Erk members of the MAPK family (Marais,R., Cell 73:381-393; Gille, et al. (1995) EMBO J. 14:951-962; Price, M.A., et al. (1995) EMBO J. 14:2589-2601). Cumulatively, the resultsindicate that activation of the Raf/ERK pathway does not induce thecytoplasmic and nuclear changes observed with MEKK.

B. Induction of Activated MEKK Sensitizes Swiss 3T3 Cells to UV-InducedApoptosis

Because stable expression of MEKK_(COOH) appeared to inhibit clonalexpansion of Swiss 3T3 cells under G418 drug selection, clones wereisolated having inducible expression of the kinase. The Lac Switchexpression system (Stratagene) was used to control the expression ofMEKK_(COOH). Several independent clones were isolated and theirproperties analyzed in the presence or absence of IPTG-inducedexpression of MEKK_(COOH). The parental LacR+ clone expressing only theLac repressor was used as the control. Clones expressing inducibleMEKK_(COOH), as determined using an antibody recognizing the extremeCOOH-terminus of MEKK, showed a small increase in the number of cellshaving a condensed cytoplasmic and nuclear morphology relative tocontrol cells even in the absence of IPTG-induced MEKK_(COOH). This isprobably due to a basal level of MEKK_(COOH) expression in uninducedcells. The addition of IPTG to the culture media induced the expressionof MEKK_(COOH) and resulted in an increase in cells having the condensedmorphology relative to the control IPTG-treated LacR+clone. However,MEKK_(COOH) expressing cells did not growth arrest and only a fractionof the cells assumed a condensed morphology as dramatic as what wasobserved with microinjection of the MEKK_(COOH) expression plasmid. Thismaybe related to selection of cells during the cloning procedure thatadapted to a low, constitutive level of MEKK_(COOH) expression.Interestingly, no clones were isolated from a total of one hundred fiftythat were analyzed that had a significant constitutive MEKK_(COOH)expression measured by immunoblotting. In addition, the level ofMEKK_(COOH) expression following IPTG induction is certainly less thanthat achieved with nuclear microinjection.

It was found that IPTG-induced MEKK_(COOH) expression stimulated signaltransduction pathways that made the cells significantly more sensitiveto stresses that induce cell death. For example, cells expressingMEKK_(COOH) were highly sensitive to ultraviolet irradiation. Two hoursafter exposure to ultraviolet irradiation greater than 30% of theMEKK_(COOH) expressing cells became morphologically highly condensed andappeared apoptotic. In contrast, the population of uninduced cellsshowed no increase in condensed apoptotic-like cells at this time point(FIG. 16). Thus, overnight induction of MEKK_(COOH) expression modestlyincreased the basal index of morphologically condensed cells and primedthe cells for apoptosis in response to UV irradiation. The resultsindicate that MEKK-regulated signal transduction pathways enhanceapoptotic responses to external stimuli.

C. Expression of MEKK_(COOH) stimulates JNK/SAPK and the transactivationof c-Myc and Elk-1 The ability of MEKK_(COOH) but not BxBRaf expressionto induce cell death indicates that each kinase regulates differentsequential protein kinase pathways. Cells were incubated for 17 hours inthe absence or presence of IPTG and assayed for JNK/SAPK activity. Theinduction of MEKK_(COOH) expression in Swiss 3T3 cells, as predicted,stimulated JNK/SAPK activity but did not activate either ERK or p38/Hog1activity as shown in FIGS. 17 and 18. The results indicate thatinduction of MEKK_(COOH) results in the activation of JNK/SAPK whichphosphorylates GST-c-Jun. Because known substrates for JNK/SAPK aretranscription factors, we assayed MEKK_(COOH) inducible clones fortransactivation of specific gene transcription. Chimeric transcriptionfactors having the Gal4 DNA binding domain and the transactivationdomain of c-Myc, Elk-1 or c-Jun were used for assay of MEKK_(COOH)signaling using a Gal4 promoter-luciferase reporter gene (Hibi et al.supra; Sadowski, I et al. (1988) Nature 335:563-564; Gupta et al. supra;Marais et al. supra.). Surprisingly, IPTG-induced stable expression ofMEKK_(COOH) markedly activated the transactivation function of c-Myc andElk-1 but had little effect on Gal4/Jun activity as illustrated in FIG.18. This result was unexpected since MEKK_(COOH) transient expressionstimulated Gal4/Jun activity, indicating that transient expression ofMEKK_(COOH) was capable of transactivating c-Jun function in Swiss 3T3cells. In addition, the JNK/SAPK activity stimulated by IPTG-inductionof MEKK_(COOH) correlated with the characterized JNK/SAPK enzyme byfractionation on Mono Q FPLC. Thus, MEKK_(COOH) expression in stableclones achieved with IPTG-induction selectively regulated Gal4/Myc andGal4/Elk-1 but not Gal4/Jun even though JNK/SAPK was activated.

The failure of IPTG-induced MEKK_(COOH) expression to activate Gal4/Junmay be related to the multiple c-Jun NH2-terminal phosphorylation sitesinvolved in regulating c-Jun transactivation. Serines 63 and 73 andthreonines 91 and 93 are apparent regulatory phosphorylation sites inc-Jun (Kyriakis et al. (1994) Nature 369:156-160; Derijard, B et al.(1994) Cell 76:1025-1037; Pulverer et al. (1991) Nature 353:670-674;Papavassiliou, et al. (1995) EMBO J. 14:2014-2019). Both clusters areproposed to be sites of phosphorylation for ERKs and JNK/SAPKs(Papavassiliou et al. supra). Transient transfection of MEKK_(COOH)activates JNK/SAPK but also activates ERKs (Lange-Carter et al. supra).In contrast IPTG-induction of MEKK_(COOH) results in the activation ofJNK/SAPK but not Erks. The difference in regulation of c-Juntransactivation may be related to the differential phosphorylation ofthese sites by JNK/SAPK and ERKs. Expression of activated Raf in Swiss3T3 cells stimulated Elk-1 transactivation, but not c-Myc or c-Juntransactivation. This result indicates that Elk-1 transactivation alonedoes not mediate the cell death response in fibroblasts observed withMEKK_(COOH). Cumulatively, the findings demonstrate that induction ofMEKK_(COOH) expression enhances cell death independent of ERK, p38/Hog-1or c-Jun transactivation in Swiss 3T3 cells and may involve c-Myctransactivation.

D. Inhibitory JNK/SAPK does not Attenuate MEKK Stimulated c-MycTransactivation or Cell Condensation

To determine if JNK/SAPK activation was required for c-Myctransactivation in response to MEKK_(COOH), Gal4/Myc activation wasassayed in the presence or absence of JNK/SAPK(APF). The results areshown in FIG. 19. The JNK/SAPK(APF) was used as a competitive inhibitorof JNK/SAPK for activation by the immediate upstream JNK kinase/SEK-1enzyme (Kyriakis et al. supra; Sluss, et al (1994). Mol. Cell. Biol.14:8376-8384; Lin et al (1994) Science 268:286-290; Sanchez et al.(1994) Nature 372:794-800). In transient transfection assays, expressionof JNK/SAPK(APF) inhibited approximately 65% of the Gal4/Jun activationin response to MEKK_(COOH). In contrast, expression of JNK/SAPK(APF) hadno effect on MEKK_(COOH) activation of Gal4/Myc induction of luciferaseactivity. Thus, c-Jun transactivation appears to be independent of theMEKK_(COOH) stimulated pathway leading to c-Myc transactivation.Similarly, JNK/SAPK activation can be significantly inhibited with noeffect on c-Myc transactivation.

The cell death response to MEKK_(COOH) also appeared to be largelyindependent of JNK/SAPK. In several experiments, expression ofJNK/SAPK(APF) alone had no demonstrative effect on Swiss 3T3 cells. Theexpressed JNK/SAPK(APF) was localized in both the cytoplasm and nucleuswhile β-gal expression was restricted to the cytoplasm. Co-expression ofJNK/SAPK(APF) with MEKK_(COOH) did not block MEKK_(COOH)-inducedcytoplasmic shrinkage and cellular condensation. A 20-fold lowerconcentration of MEKK_(COOH) still induced the cytoplasmic shrinkagecharacteristic of apoptosis in microinjected Swiss 3T3 cells.Co-microinjection of a 30-fold greater concentration of JNK/SAPK(APF)plasmid relative to the MEKK_(COOH) plasmid did not affect theMEKK_(COOH)-mediated cell death response. Cells undergoing a dramaticcytoplasmic shrinkage. Because of the low amount of MEKK_(COOH)expression plasmid used, the cell condensation response was slower inonset. The percentage of MEKK_(COOH) microinjected cells committed tocytoplasmic shrinkage and cellular condensation and the timing of thisresponse was the same in the presence or absence of JNK/SAPK(APF). Inaddition, the competitive inhibitory mutant K116RJNKK/SEK-1, the kinaseimmediately upstream of JNK/SAPK which phosphorylates and activatesJNK/SAPK (Lin et al supra; Sanchez, I (1994) Nature 372:794-800) alsounable to attenuate MEKK_(COOH) induced cell death. Expression ofJNK/SAPK(APF) or K116RJNKK/SEK-1 alone had no measurable effect on themorphology of Swiss 3T3 cells (not shown). Thus, MEKK_(COOH) inducescell death via the regulation of signal pathways that appear largelyindependent of JNK/SAPK regulation and c-Jun transactivation. Finally,BxBRaf neither induced cell death nor activated c-Myc (not shown)indicating that MEKK_(COOH)-regulated responses were not mediated by theErk1 and 2 proteins (p42/p44 MAP kinases), consistent with the lack ofERK activation in the inducible MEKK_(COOH) Swiss 3T3 cells.

These results demonstrate, for the first time, a role for MEKK inmediating a cell death response characteristic of apoptosis. Receptorssuch as the cytotoxic TNFα receptor and Fas must be capable ofregulating signal transduction pathways controlling cytoplasmic andnuclear events involved in apoptosis. The enhanced apoptosis toultraviolet irradiation observed with MEKK_(COOH) expression in Swiss3T3 cells indicates that MEKK-regulated signal transduction pathwaysintegrate with the apoptotic response system. MEKK_(COOH) expressingcells have a higher basal apoptotic index and are primed to undergoapoptosis in response to a stress stimulation. The short time requiredto observe the enhance apoptosis (2 hr) suggests that cell cycletraverse, DNA synthesis, or significant transcription/translation is notrequired for the enhanced cell death in response to ultravioletirradiation in cells expressing MEKK_(COOH). This finding is strikingand suggests that genetic or pharmacological manipulation of MEKKactivity could be used to sensitize cells to irradiation-induced death.

The ability to dissociate c-Jun transactivation fromMEKK_(COOH)-stimulated cell death argues that the JNK/SAPK activityachieved in the inducible Swiss 3T3 cell clones is insufficient alone toactivate c-Jun transactivation or induce cell death. It is more likelythat the JNK/SAPK activity we have measured is involved in stimulating aprotective program in response to potentially lethal stimuli aspreviously proposed (Devary, Y et al. (1992) Cell 71:1081-1091).Protective responses could involve changes in metabolism or alterationsin the activity of proteins such as Bc 1-2 (Gottschalk, A. R., et al.(1994) Proc. Natl. Acad. Sci. USA 91:7350-7354; Korsmeyer, S. J. (1992)Immunol. Today 13:285-290). This prediction is consistent with theactivation of JNK/SAPK mediated by CD40 ligation in B cells whichprotects against rather than stimulates apoptosis (Sumimoto, S. I., etal. (1994) J. Immunol. 163:2488-2496; Tsubata, T. et al. (1993) Nature364:645-648).

Recently, it was shown that dominant negative c-Jun could protectneurons from serum deprivation-induced apoptosis (Ham, J. et al. (1995)Neuron 14:927-939). It was proposed that the dominant negative cJuninactivated c-Jun and prevented an attempt by the post mitotic neuronsto enter an abortive cell cycle progression that triggered a cell deathprogram. Thus, dominant negative c-Jun was believed to maintain theneurons in stringent growth arrest. At first glance, the protectiveeffect of dominant negative c-Jun seems contradictory to our resultsthat JNK/SAPK and c-Jun transactivation are not involved in MEKK-inducedcell death. Our results demonstrate that the dramatic cytoplasmicshrinkage, nuclear condensation and onset of cell death induced byMEKK_(COOH) are largely independent of JNK or c-Jun transactivation.Importantly, MEKK_(COOH)-induced cell death occurs in high serum wheregrowth factor and cytokine stimulation of the cells is normal. We havealso determined that expression of MEKK_(COOH) in Swiss 3T3 cells doesnot significantly inhibit or alter cell cycle progression. Thus, anabnormal cell cycle event that may occur with serum deprivation does notappear to account for MEKK-induced cell death.

Expression of MEKK_(COOH) increased the transactivation of c-Myc andElk-1 in Swiss 3T3 cells. c-Myc has been shown to be required forapoptosis in lymphocytes (Fanidi, A et al. (1994) Nature 359:554-556;Janicke, R. U. et al (1994) Mol. Cell. Biol. 14, 5661-5670; Shi et al.(1992) Science 257:212-214), to induce apoptosis when overexpressed ingrowth factor-deprived fibroblasts (Harrington, E. A. et al. (1994) EMBOJ. 13:3286-3295); Askew, D. W., et al. (1991) Oncogene 6:1915-1922;Evan, G. I. et al. (1992) Cell 69:119-128), and to enhance TNF-mediatedapoptosis (Klefstrom, J., et al. (1994) EMBO J. 13:5442-5450). Therequirement of c-Myc for apoptosis is not understood mechanistically,but c-Myc is proposed to transcriptionally activate an apoptotic pathway(Harrington, E. A. et al. (1994) EMBO J. 13:3286-3295); Askew et al.supra; Evan et al. supra, Janicke et al. supra; Shi et al. supra). Theactivation of Elk-1 by MEKK_(COOH) induction in Swiss 3T3 cellscorrelates best with the stimulation of JNK/SAPK. Recently, it was foundthat JNK/SAPK in addition to Erks phosphorylated and activated Elk-1consistent with our findings (Whitmarsh, A. J. et al. (1995) Science269:403-407). In contrast, we demonstrate that c-Jun is notsignificantly activated in MEKK_(COOH) expressing cells. These findingsare provocative because they indicate that MEKK-stimulated JNK/SAPKactivation preferentially regulated Elk-1 and not c-Jun. A second signalin addition to JNK/SAPK may be required for c-Jun transactivation incells (Papavassiliou, A. G., et al. (1995) EMBO J. 14:2014-2019). Theredoes not seem to be a proposed role for Elk-1 in inducing an apoptoticresponse, but serum deprivation-induced apoptosis of Swiss 3T3 cellsresults in the increased expression of early cell cycle genes consistentwith an increased SRF/SRE activity associated with elevated Elk-1activity (Pandey, S. and Wang, E. (1995) J. Cell. Biochem. 58:135-150).The induction of apoptosis in several cell types does not appear torequire transcription, but the use of inducible cell lines and plasmidmicroinjection experiments do not facilitate testing whether MEKK_(COOH)can induce cell death in the absence of transcription. In cells wheretranscription is not necessary for the induction of apoptosis it islikely that proteins required for apoptosis are already expressed andmay be post translationally regulated by sequential protein kinasepathways involving MEKK. For example, the phosphorylation of nuclearproteins could alter their activity independent of transcription andcontribute to a cell death response.

In Jurkat cells, a human T cell line, Fas-induced apoptosis has beenproposed to involve a ceramide stimulated, Ras-dependent signalingpathway (Gulbins, E., et al. (1995) Immunity 2:34351). We recentlydemonstrated that MEKK activity can be stimulated by Ras and that MEKK1physically binds to Ras in a GTP-dependent manner (Russell, M. et al.(1995) J. Biol. Chem. 270:11757-11760; Winston, B. W., et al. (1995)Proc. Natl. Acad. Sci. USA (1995) 92:1614-1618). The ability of MEKK toregulate an apoptotic-like cell death response suggests it is acandidate component for the ceramide regulated apoptotic pathway.

The importance of our observations describing the involvement of MEKKregulated sequential protein kinase pathways in physiologically relevantsignaling leading to cell death is supported by several findings. First,MEKK_(COOH) induces or enhances a cell death response in the presence of10% calf serum, indicating that growth factor deprivation is not aprerequisite for MEKK-induced cell death. This is similar to TNFα, Fasand ceramide-mediated apoptosis which proceeds in high serum. Thus, theinvolvement of MEKK in cell death responses is not simply to activate asubset of growth factor stimulated signaling events causing an abortedcell cycle-induced apoptosis that would normally be prevented by serumfactors. Second, the enhanced cell death to ultraviolet irradiationindicates that expression of MEKK_(COOH) may activate signals thatpotentiate stresses to the cell. This finding indicates thatMEKK-regulated signal transduction pathways integrate with cellularresponses involved in mediating apoptosis, that ultraviolet irradiationlikely activates additional pathways and that MEKK_(COOH)-mediatedsignaling synergizes with the ultraviolet response to accelerateapoptosis. Third, MEKK stimulated sequential protein kinase pathwaysindependent of ERK, JNK/SAPK, p38/Hog1 and c-Jun transactivation thatcan stimulate c-Myc transactivation. These results indicate thatMEKK-regulated pathways traverse the cytoplasm to regulate as yetundefined protein kinases that activate cMyc in the nucleus. Theregulation of c-Myc activity is a unique function of MEKK signaling andone that we postulate is likely to contribute to the cell deathresponse. Serum deprivation significantly induces JNK/SAPK activation inseveral cell types including Swiss 3T3 cells. Similarly, TNF αstimulates a JNK/SAPK pathway (Minden et al. (1994) Science266:1719-1723) and we have recently demonstrated TNFα stimulation ofMEKK activity in mouse macrophages (Winston et al. supra). c-Mycoverexpression has been shown to enhance TNFα receptor stimulation ofapoptosis (White et al. (1992) Mol. Cell. Biol. 12:2570-2580). Thesefindings are consistent with a linkage between TNFα receptor signaling,MEKK and c-Myc. Cumulatively, the findings define MEKK as a potentiallyimportant component in the regulation of signal transduction pathwaysinvolved in apoptosis.

Example 21 This Example Illustrates that TNF and Expression of MEKK1COOHSynergize to Induce Apoptosis in Cells

Control L929 fibroblasts (4.1 LAC1), fibroblasts expressing MEKK1_(COOH)domain (15.10 LAC1), or fibroblasts expressing the kinase inactivemutant of MEKK1_(COOH) (41.112 LAC1) using the Lac Switch expressionsystem described in Example 19, were treated with TNF in the presence orabsence of IPTG and the percentage of apoptotic cells was calculated. Asshown in FIG. 20, approximately 20% of control L929 cells becameapoptotic upon TNF exposure either in the presence and absence of IPTG.In L929 cells expressing the MEKK1COOH domain, exposure to TNF and IPTGincreased the percentage of apoptotic cells to 40%, approximately a2-fold increase. In L929 cells expressing the MEK kinase inactivemutant, exposure to TNF did not increase the level of apoptotic cellsabove levels seen in controls, in fact the percentage of apoptotic cellswas slightly decreased in cells exposed to both TNF and IPTG.

Example 22 This Example Describes Regulation of MAPK Activity by BothMEKK and Raf Protein

COS cells were prepared using the method described in Example 3. Inaddition, COS cells were transfected with the pCVMV5 Raf construct (1μg: Raf). FPLC MONO Q ion-exchange column fractions were prepared asdescribed in Example 3 and assayed for MAPK activity according to themethod described in Heasley et al., ibid.

Referring to FIG. 21, both MEKK and Raf overexpression in COS 1 cellsresulted in similar levels of stimulation of MAPK activity over basallevels.

Example 23 This Example Demonstrates the Ability of Cos Cell-ExpressedMEKK1 Proteins to Bind to GST-Ras^(V12)

COS cells were transiently transfected by the DEAE-dextran protocol asgenerally described in Example 3. Cos cells were transfected with: (1)p-MEKK1 containing a nucleic acid molecule encoding MEKK1 as describedin Lange-Carter et al. (Science 260:315-319, 1994); (2) p-MEKK_(NH2)containing a nucleic acid molecule that encodes a 858 base pairPvull(682)-Ncol(1541) restriction digest fragment of the amino terminusof MEKK1 ligated into pCMV5; (3) p-MEKK_(COOH) containing a nucleic acidmolecule that encodes a 1435 base pair Ncol(1541)-Sspl(2976) restrictiondigest fragment that includes the entire kinase domain of MEKK1 ligatedinto pCMV5; (4) pCMV5 without insert; or (5) p-C4Raf containing anucleic acid molecule that encodes the amino terminus of Raf-1 ligatedinto pCMV5. COS cells expressing the various MEKK1 proteins wereselected by the method described in COS cells expressing the variousMEKK1 proteins were lysed in EB (1% Triton X-100, 10 mM Tris HCl [pH7.4], 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 0.1% bovine serum albumin, 0.2U/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride and 2 mM Na₃ VO₄).The lysates were separated into two equal parts for separate bindingreactions. Half of the lysate was incubated with GST agarose (1.5 μg)while half of the lysate was incubated with GST-Ras^(V12) agarose (1.5μg) (purchased from UBI) for 1 hr at 4° C. The GST-Ras^(V12) waspreincubated at 30° C. for 30 min with 1 mM nucleotide (GDP or GTPγS).The nucleotide binding reaction was stopped by adding MgCl₂ to a finalconcentration of 20 mM. After the 1 hr binding reaction the agarosebeads were pelleted at 2000 rpm for 2 min and washed 3 times withPBS+1.0% Triton X-100. The washed agarose beads were boiled in LaemmliSDS sample buffer and the proteins resolved by SDS polyacrylamide gelelectrophoresis. Proteins were transferred onto nitrocellulose forimmunoblotting with antibodies specific for an NH₂ terminal fusionprotein (described in Example 1) or a COOH terminal peptide (describedin Example 1). C4Raf binding was detected using an antibody specific forRaf described in Example 8.

Initial immunoblotting results using anti-Raf antibodies demonstratedthat C4Raf bound to GST-Ras^(V12)(GTPγS) agarose but not to the GSTagarose control. Additionally, no Raf immunoreactive proteins weredetected bound to Ras from COS cells transfected with pCMV5. Theseresults indicated that the Ras binding assay was functional.

Immunoblotting results using anti-MEKK antibodies indicate that proteinencoded by p-MEKK1 (MEKK1) transiently expressed in COS cells wascapable of binding GST-Ras^(V12) in a GTP dependent manner. MEKK1 fromCOS cell lysates bound to GST-Ras^(V12)(GTPγS), while little binding toGST-Ras^(V12)(GDP) was detectable. With the conditions used, MEKK1binding to GST-Ras^(V12)(GTPγS) was at least 5-fold greater than thebinding to GST-Ras^(V12)(GDP). No detectable MEKK1 was bound to GST.

The domain critical for the binding of MEKK1 to Ras was then identified.The protein encoded by p-MEKK_(COOH) (MEKK_(COOH)) bound toGST-Ras^(V12) in a GTP dependent manner. Little MEKK_(COOH) bound toGST-Ras^(V12)(GDP). No detectable MEKK_(COOH) was bound to GST. Inaddition, when protein encoded by p-MEKK_(NH2) (MEKK_(NH2)) wasexpressed in COS cells, no binding to Ras was detected. In contrast tothe ability of Raf-1 to bind to Ras through its amino terminus,MEKK_(NH2) failed to bind GST-Ras^(V12)(GTPγS) even though the proteinwas expressed to similar levels as MEKK1 in the same experiment. Thus,GST-Ras^(V12) binds to MEKK1 at a site located within the COOH-terminalcatalytic domain of MEKK1.

Example 24 This Example Demonstrates the Ability of Purified RecombinantMEKK1 Proteins to Bind Directly to GST-Ras^(V12)

A construct encoding the kinase domain of a Rat MEKK1 cDNA (95%identical to mouse MEKK1) with a N-terminal hexahistidine tag (referredto herein as MEKK_(COOH)-His; provided by Dr. Melanie Cobb, Departmentof Pharmacology, University of Texas Southwestern Medical School,Dallas, Tex.) was expressed in bacteria and soluble active enzyme waspurified on Ni₂+-NTA agarose according to the method generally describedin Gardner et al. (Methods of Enzymology 238:258-270, 1994) Purifiedrecombinant MEKK_(COOH)-His was incubated with either GST orGST-Ras^(V12) in PAN buffer (10 mM PIPES [pH 7.0], 100 mM NaCl, 0.2 U/mlaprotinin) for 1 hr at 4° C. The agarose beads were pelleted and washed3 times in PAN buffer. The washed agarose beads were then incubated inkinase buffer (20 mM PIPES [pH 7.0], 10 mM MnCl₂, 40 μCi[γ³²P]ATP, 20μg/ml aprotinin) containing 100 ng recombinant kinase inactive MEK1 assubstrate in a final volume of 150 μl, at 30° C. for 20 min. To test thedirect interaction of MEKK1 with the effector domain of Ras, sampleswere prepared by pre-incubating the agarose beads with either 100 μM ofRas peptide consisting of residues 17-42 of H-Ras or 100 μM of Rascontrol peptide ([D-Arg¹,D-Phe⁵,DTrp^(7,9),Leu¹¹] substance P peptidefor 1 hr at 4° C. prior to addition of the MEK1 substrate. A controlreaction containing wild-type MEKK1 which autophosphorylates, served asa marker for the MEKK1 substrate. Reactions were terminated by additionof 5× Laemmlei SDS sample buffer, boiled and resolved by SDS-PAGE.

The results indicate that there was direct binding of Ras-GTPγS topurified MEKK_(COOH)-His as measured by the increased phosphorylation ofKM MEK1 using GST-Ras^(V12)(GTPγS) beads incubated with recombinantMEKK_(COOH)-His. The interaction between Ras and MEKK_(COOH)-His was GTPdependent because essentially no KM MEK1 phosphorylation could bedetected with GST-Ras^(V12)(GDP) beads incubated with recombinantMEKK_(COOH).

The results indicate that the presence of Ras effector peptide preventedthe binding of GST-Ras^(V12)(GTPγS) agarose to MEKK_(COOH)-His, therebypreventing the phosphorylation of KM MEK1 substrate present in thesample. MEKK_(COOH)-His was able to bind to GST-Ras^(V12)(GTPγS) in thepresence of buffer alone or in the presence of a control peptide([D-Arg¹,D-Phe⁵,D-Trp^(7,9),Leu¹¹] substance P peptide), resulting inthe phosphorylation of KM MEK1 substrate.

Taken together, the results described in Examples 22 and 23 demonstratethat MEKK1 is a Ras effector and selectively binds to Ras in a GTPdependent manner. In addition, the binding of MEKK1 to Ras in vitro isdirect and occurs via the COOH terminal region of MEKK1 that encodes thecatalytic kinase domain.

Example 25 This Example Demonstrates the Cloning of MEKK4.1 and MEKK4.2,a Splicing Variant of MEKK4

The degenerate primers GA(A or G)(C or T)TIATGGCIGTIAMINO ACIDS(A orG)CA (sense) and TTIGCICC(T or C)TTIAT(A or G)TCIC(G or T_)(A or G)TG(antisense) were used in a polymerase chain reaction (PCR) using firststrand cDNA generated from polyadenylated RNA prepared from NIH 3T3cells. The PCR reaction involved 30 cycles (1 minute, 94° C./2 minutes,52° C./3 minutes, 72° C.) followed by a 10 minute cycle at 72° C. A bandof approximately 300 bp was recovered from the PCR mixture and theproducts cloned into pGEM-T (Promega). The PCR cDNA products weresequenced and compared to the MEKK1 sequence. A unique cDNA sequencehaving significant homology to MEKK1 cDNA was identified and used toscreen an oligo dT primed mouse brain cDNA library (Stratagene). The Xphage library was plated and DNA from plaques transferred to hybond-Nfilters (Amersham) followed by UV-crosslinking of DNA to the filters.Filters were pre-hybridized for 2 hours and then hybridized overnight in0.5 M Na₂H₂PO₄ (pH 7.2), 10% bovine serum albumin, 1 mM EDTA, 7% SDS at68° C. Filters were washed 2× at 42° C. with 2×SSC, 1× with 1×SSC and 1×with 0.5×SSC containing 0.1% SDS (1×SSC is 0.15 M NaCl, 0.015M sodiumcitrate, pH 7.0). Positive hybridizing clones were purified andsequenced. To resolve GC-rich regions cDNAs were subcloned into M13vectors (New England Biolabs) and single strand DNA sequenced. In allcases both strands of DNA were sequenced. Clones were truncated at the5′-region and were therefore not full length in the coding region. Toobtain the 5′ region of MEKK4 poly RNA was isolated and primers from thepartial cDNA used for reverse transcription. cDNAs were generated usingthe RACE procedure and sequenced. The 5′ region of MEKK4 with upstreamin frame stop codons was obtained and ligated to the partial MEKK4 cDNAto give a full length MEKK4 cDNA having an open reading frame of 1597codons.

Example 26 This Example Demonstrates the Differential Expression ofMEKK4.2

RNA was isolated from the indicated tissues of a Balb/c mouse tissues.RNA was isolated from the indicated tissues of a Balb/c mouse, resolvedon an agarose gel, transferred to nitrocellulose paper and hybridizedwith ³²P-labeled MEKK4.2 cDNA probe. A single mRNA band approximately5.8 kb is hybridized with the labeled MEKK4.2 probe.

Example 27 This Example Demonstrates that the MEKK4 Kinase DomainActivates c-Jun Kinases Activity

COS cells were transfected with pCMV5 expression plasmid encoding nocDNA insert (control), full length MEKK4 or the truncated MEKK4 encodingonly the catalytic kinase domain. The truncated MEKK4 kinase domain isconsitutitively active when expressed in COS cells. The MEKK1 kinasecatalytic domain, and MEKK2 and -3 also activate the c-Jun kinasepathway (see FIG. 22).

Example 28 This Example Demonstrates that MEKK4 does not ActivateP42/P44 MAP Kinases (ERK1 and ERK2) Activity

COS cells were transfected with pCMV5 expression plasmid encoding nocDNA insert (control), full length MEKK4, the truncated MEKK4 encodingonly the catalytic kinase domain or the MEKK1 catalytic domain. TheMEKK1 catalytic domain but not the MEKK4 catalytic domain is capable ofactivating ERK1 and ERK2 (see FIG. 23).

Example 29 This Example Demonstrates that MEKK4 Interacts with Cdc42/Rac

GST fusion proteins encoding Cdc42 or Rac loaded with either GTPγs orGDP were incubated with MEKK4 using previously described methods(Russell, M. et al. (1995) J. Biol. Chem. 270:11757-11760). The sourceof MEKK4 was either from a Cos cell transient transfection or arecombinant MEKK4 protein expressed in E. coli. The recombinant MEKK4protein was truncated to express residues from 1261-1597 of the fulllength protein. A GST fusion protein of Ha-Ras was used as a control.The MEKK4 protein was incubated for 1 hr at 4° C. with either GST-Cdc42,GST-Rac or GST-Ras bound to glutathione-Sepharose beads. Each GST fusionprotein had GTPγs or GDP bound to the Cdc42, Rac or Ras moiety of thefusion protein. Following the incubation the beads were washedextensively and the bound proteins removed in SDS-Laemmli buffer andresolved by SDS-PAGE using 10% acrylamide gels. The proteins weretransferred to nitrocellulose and immunoblotted using a MEKK4 specificantibody recognizing the extreme COOH-terminus of MEKK4. MEKK4specifically bound to GST-Cdc42 and GST-Rac in the GTPγS form. The GDPbound forms of GST-Cdc42 and GST-Rac bound less than 10% of the MEKK4bound in the presence of GTPγs. MEKK4 did not bind significantly toGST-Ras in either the GTPγS or GDP bound form.

The sequence IIGQVCDTPKSYDNVHVGLRKV (residues 1306-1327) of the MEKK4sequence) was synthesized as a GST-fusion protein by standard PCRtechniques. The GST-fusion peptide bound Cdc42 and Rac in the GTPγSbound form. This fusion protein did not bind Ras using the proceduresdescribed above.

Example 30

Tumor necrosis factor a (TNFα) is a multifunctional cytokine secretedprimarily by activated monocytes (Tracy, K. J., and Cerami, A. (1993)Annu. Rev. Cell Biol. 9:317-343). It has a wide range of biologicalactivities depending upon cell type, stage of differentiation andtransformation state. TNFα acts as a growth factor for fibroblasts(Vilcek, J., et al. (1986) J. Exp. Med. 163:632-643; Victor, I., et al.(1993) J. Biol. Chem. 268:18994-18999), is cytotoxic towards certaincells and tumors (Larrick, J. W., and Wright, S. C. (1990) FASEB J.4:3215-3216), induces monocyte differentiation of the human HL-60myeloid leukemia cell line (Trinchieri, G., et al. (1986) J. Exp. Med.164:1206-1225; Kim, M., et al. (1991) J. Biol. Chem. 266:484-489),represses adipocyte (Torti, F. M., et al. (1985) Science 229:867-869)and myoblast differentiation (Miller, S. C., et al. (1988) Mol. Cell.Biol. 8:2295-2301), and mediates endotoxic shock (Tracey, K. J., et al.(1986) Science 234:470-474). The pleiotropic effects of this cytokinemake it an important mediator in processes as diverse as proliferation,differentiation and cytotoxicity.

TNFα exerts these responses by binding to two cell surface receptor, the55 kD TNFR (p55 TNFR) and the 75 kD TNFR (p75 TNFR) (Loetscher, H., etal. (1990) Cell 61:351-359; Schall, T. J., et al. (1990) Cell61:361-370; Smith, C. A., et al. (1990) Science 248:1019-1023; Heller,R. A., et al. (1990) Proc. Natl. Acad. Sci. (USA) 87:6151-6155). Thereceptors are single transmembrane spanning glycoproteins present onalmost all cells analyzed (Kull, Jr., et al. (1985) Proc. Natl. Acad.Sci. (USA) 82:5756-5760; Lewis, M., et al. (1991) Proc. Natl. Acad. Sci.(USA) 88:2830-2834). The extracellular domain of the p55 TNFR ishomologous to the extracellular domains of the low affinity nerve growthfactor receptor, the Fas/APO1 receptor, CD40, OX40, and CD27. The p55TNFR and Fas share a 65 residue homology region in the cytplasmicdomains (Tartaglia, L. A., and Goeddel, D. V. (1992) Immunol. Today13:151-153; Smith, C. A., et al. (1994) Cell 76:959-962) which deletionstudies have implicated in the TNFα signaling cascade leading toapoptosis (Itoh, N., and Nagata, S. (1993) J. Biol. Chem.268:10932-10937; Tartaglia, L. A., et al. (1993) Cell 74:845-853). Mostof the known TNFα responses occur by activation of the p55 TNFR.However, thymocyte proliferation is associated with p75 TNFR andeytotoxicity may be a function of p75 TNFR acting alone or in concertwith the p55 TNFR (Heller, R. A., et al. (1992) Cell 70:47-56).

Apoptosis involves the activation of a specific suicide program within acell. It occurs when a cell initiates a series of biochemical andmorphological events which result in nuclear disintegration and eventualfragmentation of the dying cell into a cluster of membrane-boundapoptotic bodies (Kerr, J., Wyllie, A., and Currie, A. (1972) Br. J.Cancer 26:239-257). Apoptosis is responsible for such diverse activitiesas the elimination of cells during normal embryological development anddetermination of the immune receptor repertoire (Raff, M. C. (1992)Nature 356:297-300; Krammer, P. H., et al. (1994) Curr. Opin. inImmunol. 6:279-289; Green, D. R., and Scott, D. W. (1994) Curr. Opin. inImmunol. 6:476-487)). Apoptosis can be triggered in multiple ways, butit is not yet known whether different inducers of apoptosis have acommon pathway or whether there are multiple pathways with perhaps somecommon components.

In many peptide-hormone receptor systems signal transduction to thenucleus involves the sequential activation of protein kinases. Theextracellular response kinase (ERK) group of mitogen-activated proteinkinases (p42 and p44 MAPK) are activated by growth factors via a Ras/Rafdependent signal transduction pathway (Davis, R. J. (1993) J. Biol.Chem. 268:14553-14556; Cano, E. and Mahadevan, L. (1995) Trends Biochem.Sci. 20:117-122). In contrast, the JNK/SAPK (Jun kinase/stress-activatedprotein kinase) members of MAPKs are activated by proinflammatorycytokines and environmental stresses (Devary, et. al. (1992) Cell71:1081-1091; Hibi, M., et al. (1993) Genes & Development 7:2135-2148;Sluss, H., et al. (1994) Mol. Cell. Biol. 14:8376-8384; Kyriakas, J. M.,et al. (1994) Nature 369:156-160; Minden, A., et al. (1994) Mol. Cell.Biol. 14:6683-6688).

TNFα has been shown to initiate apoptotic cell death and DNAfragmentation in several mammalian cell lines, including the murinefibrosarcoma cell line L929 (Kyprianou, N., et al. (1991) J. Natl.Cancer Inst. 83:346-350; Feshel, K., et al. (1991) Am. J. Pathol.139:251-254). RNFa also has been shown to activate p42/p44 MAPK in thiscell line (Van Lint, J., et al. (1992) J. Biol. Chem. 267:25916-25921).Recently JNKs were shown to be activated by TNFα (Westwick, J., et al.(1994) J. Biol. Chem. 269:26396-6401) and activation of the JNK pathwaycorrelated with enhanced apoptosis of PC12 cells in response to trophicfactor deprivation (Xia, Z., et al. (1995) Science 270:1326-1331). Wehave characterized the regulation of MAPKs and JNKs in L929 cellschallenged with TNFα and basic fibroblast growth factor (bFGF). We showthat TNFα preferentially activates JNK in L929, cells; but that JNKactivation is not sufficient to induce apoptosis, since bFGF mediates aprotective effect against TNFα mediated apoptosis without affecting JNKactivation. Furthermore, our data indicate that p42/p44 MAPK activationis required for bFGF supression of TNFα mediated apoptosis.

Materials and Methods

Cell lines and culture. L929 cells (ATCC CCL1 were maintained inDulbecco's modified Eagle's medium with 5% newborn calf serum and 5%bovine calf serum (BCS) supplemented with 100 ug/ml streptomycin and 100U/ml penicillin. The cells were grown in 10 cm dishes at 37° C. in 7.5%CO2. Cells were made quiescent where indicated by incubation inDulbecco's modified Eagle's medium and 0.1% bovine serum albumin for 24h. Recombinant murine TNFα and recombinant human bFGF (147aa) were fromR&D Systems, Minneapolis, Minn. Cells were pretreated where indicatedwith the MEK-1 inhibitor PD#098059 (Parke-Davis Pharmaceutical Corp. AnnArbor, Mich.) for 1 h at 37° C. Cells were stimulated by incubation withthe indicated cytokine or growth factor for various times at 37° C.Cells were stimulated by incubation with the indicated cytokine orgrowth factor for various times at 37° C. Stimulation was stopped byrinsing the plates twice with ice cold phosphate buffered saline (PBS)and lysing the cells in the appropriate lysis buffer. Cells were scrapedfrom the plates and nuclei were pelleted for 10 min at 14,000 RPM in amicrocentrifuge.JNK assay. JNK activity was measured using a solid state kinase assay inwhich glutathione S-transferase-c-Jun (1-79) (GST-JUN) cound toglutathione-Sepharose 4B beads was used to affinity purify JNK and thenJNK activity was measured in an in vitro kinase assay using thesepharose bound GST-Jun as a substrate (Hibi, M., et al. (1993) Genes &Development 7:2135-2148). Stimulated or unstimulated cells were lysed in0.5% Nonidet P-40, 20 mM HEPES pH 7.2, 100 mM NaCl, 2 mM dithiothreitol,1 mM EDTA, 1.0 mM phenylmethylsulfonylfluoride, 1 μg/ml aprotinin andthe nuclei pelleted. Lysates were normalized for protein content. JNKwas affinity purified from 50-100 μg of cell lysate by the addition of10 ul of GST-Jun sepharose slurry (2 μg GST-Jun). Binding to GST-Junefficiently isolates the two major forms of JNK (p45 and p55) and underthe conditions used JNK isolation was linear for 10-250 μg of celllysate. The lysates were rotated at 4° C. for 1-3 h. Beads were washedtwice in lysis buffer and then twice in PAN (10 mM PIPES, pH 7.0, 100 mMNaCl, 21 μg/ml aprotinin). Kinase reactions were carried out at 30° C.for 15 min in 20 mM Hepes pH 7.2, 20 mM β-glycerophosphate, 10 mMp-nitrophenyl phosphate, 10 mM MgCl₂, 1 mM dithiothreitol, 50 μm sodiumvanadate, 10 μCi γ³²P-ATP 4300 Ci/mmole. The kinase reaction was linearfrom 0-30 min.MAPK Assay MAPK activity was measured exactly as described previously(Gardner, A. M., et al. (1994) Meth. Enzymol. 238:258-270) with theexception that MonoQ FPLC fractionation was replaced by step elutionfrom a DEAE-Sephacel column using 0.5 M NaCl in loading buffer. Theeluate was assayed in triplicate using the epidermal growth factorreceptor 662-681 peptide (EGFR₆₆₂₋₆₈₁) as a selective substrate for MAPKactivity (Heasley, L. E., et al. (1994) American Journal of Physiology(Renal Fluid Electrolyte Physiol. 36) 267:F366-F373).Raf Activation Assay Cells were serum starved and challenged in thepresence or absence of the appropriate cytokine or growth factors, asdescribed above. Cells were lysed by scraping in ice cold RIPA buffer(50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.1% SDS, 0.5% sodiumdeoxycholate, 1.0% Triton X-100, 10 mM sodium pyrophosphate, 25 mMβ-glycerophosphate, 2 mM sodium vanadate, 2.1 μg/ml aprotinin) and thenuclei were pelletted. The supernatants were normalized for proteincontent and precleared with protein A Sepharose prior toimmunoprecipitation with rabbit antiserum to the C terminus of C-Raf,rabbit anti-serum to A-Raf or rabbit antiserum to B-Raf (Santa CruzBiotech., Santa Cruz, Calif.) and protein A Sepharose for 2-3 hr at 4°C. The beads were washed twice with ice cold RIPA and twice with PAN. Athird of the immunoprecipitate was diluted with SDS sample buffer andused for immunoblot analysis. The remainder was resuspended in kinasebuffer (20 mM Pipes pH 7.0, 10 mM MnCl₂, 150 ng kinase-inactive MEK-1,30 μCi γ³²P-ATP and 20 μg/ml aprotinin) in a final volume of 40 μl for30 min at 30° C. Wild-type recombinant MEK-1 was autophosphorylated inparallel as a marker. Reactions were terminated by the addition of 12.5μl 5×SDS sample buffer, boiled, and subjected to SDS-PAGE andautoradiography.Neutral Red Assay Uptake of the dye neutral red was used as one measureof cell viability following cytokine or growth factor treatment (Finter,N. B. (1969) J. Gen Virol. 5:419-427). 1.5×10⁴-2.5×10⁵ L929 cells/wellwere plated in 12 well tissue culture dishes in 1.25 ml of media. Cellswere treated for 15-20 hr with various concentrations of TNFα and/orbFGF. 2.5 μl of 1% neutral red was added to the wells and incubated for2 hr at 37° C. PBS. The neutral red was extracted with 1.0 ml of 50%ethanol, 50 mM Na-citrate pH 4.2 and absorbency was measured at 540 mM.Propidium iodide staining Cells were plated on glass chamber slides(Nunc, Naperville, Ill.) at a concentration of 0.2-0.6×10⁵ cells/ml. Rasexpression was induced with 5 mM IPTG in Dulbecco's modified Eagle'smedium with 0.1% BCS for 8-12 hr. Cells were exposed to TNFα (5 ng/ml)and/or bFGF (500 pg/ml) in Dulbecco's modified Eagle's medium with 0.1%BCS for 16 hr. The parental LACI expressing cell line (see below) wasused as a control. Cells were washed twice in PBS, fixed inacetone:methanol (1:1)-20° C. for 5 min, air dried, washed twice in PBS,stained with 1 μg/ml propidium iodide (PI) in PBS for 20 min, washed inPBS, washed in H₂O and mounted in 25% glycerol/PBS. PI fluorescence wasobserved using a Nikon inverted microscope equipped with epifluorescenceand a 580 mm filter. Images were analyzed using IP lab.Cell transfections L929 cells were transfected by CaPO₄ (Ausubel, F.(1994) Current Protocols in Molecular Biology Vol. 1, pp. 9.1.1-9.1.4,John Wiley & Sons, Inc., New York) with the vector 3′SS (Stratagene, LaJolla, Calif.) expressing the LACI repressor. Stable clones wereselected in 200 μg/ml hygromycin (Calbiochem, La Jolla, Calif.) andscreened for LACI expression by indirect immunofluorescence using rabbitanti-sera to LACI (Stratagene, La Jolla, Calif.) and FITC-donkeyanti-rabbit. One clone expressing a high level of nuclear LACI was thentransfected with hemaglutinin (HA)-tagged inhibitory N17 (Feig, L. A.and Cooper, G. M. (1988) Mol. Cell. Biol. 8:3235-3243) Ras or activatedV12 Ras (Tobin, C., et al. (1982) Nature 300:143-148; Reddy, E. P., etal. (1982) Nature 300:149-152); Taparowsky, E., Suard, Y., Fassano, D.,Simiger, K., Goldfarb, M., and Wigler, M. (1982) Nature 300:149-152)cloned into the LACI repressible pOPRSVI vector. Stable clones wereselected in 500 μg/ml G418 and screened for inducible expression ofHA-Ras by immunoblotting. Incubation in 5 mMisopropyl-1-thio-β-D-galactopyranoside (IPTG) for 8-24 hr was used toinduce Ras expression. Several independent, inducible N17 Ras or V12 Rasclones were isolated and two each were chosen for further analysis.Immunoblotting 100 μg of cell lysate was fractionated by SDS PAGE (12.5%acrylamide) and blotted to nitrocellulose in 10 mM CAPS, pH 11, 20% MeOHusing a Transphor apparatus (Hoeffer, San Diego, Calif.) for 1 hr at 1amp. Blots were blocked in 5% powdered milk in Tris-HCl, pH 7.5 bufferedsaline. Ras was detected with Y-13259 anti-Ras monoclonal antibody(Fruth, M. E., Davis, L. J., Fleurdelys, B., and Skolnick, E. M. (1982)J. Virol. 43:294-304) followed by enhanced chemiluminescence (Amersham,Chicago, Ill.) using HRP-anti-mouse IgG (BioRad, Richmond, Calif.).Quantitation of data PhosphorImager analysis of phosphorylated proteinsprovided a quantitative measure of kinase activation in arbitraryphosphorimaging units. Statistical analysis was performed using the JMPprogram and the method of Tukey & Kramer was used to determinestatistical differences.

Results

bFGF protects L929 from TNF α-mediated apoptosis TNFα activates a celldeath program resulting in the apoptosis of L929 cells (Feshel, K.,Kolb-Bachofen, V., and Kolb, H. (1991) Am. J. Pathol. 139:251-254). FIG.24A shows that treatment of L929 cells overnight with TNFα resulted insubstantial cell death using the neutral red assay as a measure ofviable cells (see Methods). The time course of cell death was dependenton the concentration of TNFα. Treatment with 10 ng/ml TNFα resulted ingreater than 40% of the L929 cells being apoptotic in 15 hr; 1 ng/mlTNFα required 24-48 hr to induce a similar level of L929 cell death (notshown). Serum and growth factor withdrawal induces apoptosis in severalcell systems (Oppenheim, R. W. (1991) Annu. Rev. Neurosci. 14:453-501;Kinoshita, T., et al. (1995) EMBO J. 14:266-275), indicating that growthfactors have a protective effect against apoptosis. Consistent with thisobservation was our finding that bFGF affected TNFα mediated apoptosis(FIG. 24B). Incubation of L929 cells with TNFα in the presence of bFGFwas effective at blocking TNFαα-mediated cell death. The protectiveeffect of bFGF was not simply due to an increased proliferative responseof L929 cells, because bFGF in the absence of TNFα did not measurablyincrease cell number (FIG. 24B).Regulation of JNK and MAPK by TNFα and bFGF TNFα has been previouslyshown to activate p24/p44 MAPK in L929 cells (Van Lint, J., Agostinis,P., Vandevoorde, V., Haegeman, G., Fiers, W., Merlevede, W., andVandenheede, J. (1992) J. Biol. Chem. 267:25916-25921) but recentstudies have indicated that TNFα is a potent activator of the Jun kinase(JNK) members of the MAPK family (Sluss, H., et al. (1994) Mol. Cell.Biol. 14:8376-8384; Kyriakas, J. M., et al. (1994) Nature 369:156-160;Westwick, J., Weitzel, C., Minden, A., Karin, M., and Brenner, D. (1994)J. Biol. Chem. 269:26396-6401). Analysis of the time course and doseresponse of TNFα on L929 cells demonstrated significant differences inthe activation of JNK and p42/p44 MAPK activity. Extracts fromTNFα-treated versus control L929 cells were assayed for JNK activityusing GST-c-Jun(₁₋₇₉) as substrate. TNFα induced a transient increase inJNK activity that peaked at 10-15 min and returned to two-fold abovebasal JNK activity 1-2 hr post-stimulation. Maximal JNK activation wasachieved at 1 ng/ml TNFα and 0.1 ng/ml TNFα activated JNK greater thanfour-fold. TNFα stimulation of p42/p44 MAPK activity was slightly morerapid than JNK activation, reaching maximal stimulation in 5-10 min thatreturned to near basal levels by 30 min (FIG. 25A). The dose-responsecurve for p42/p44 MAPK activation is dramatically shifted to higher TNFαconcentrations than that for JNK (FIG. 25B). Greater than 10 ng/ml TNFαwas required to stimulate p42/p44 MAPK 2-3 fold; at 1 ng/ml TNFα theMAPK activity was stimulated only 20% above basal, a concentration ofTNFα that gave maximal JNK activation. Thus, TNFα preferentiallyregulates the JNK pathway relative to p42/p44 MAPK in L929 cells. Thesefindings indicate that the localized concentration of cytokines such asTNFα will determine the selectivity and magnitude of cellular JNK andp42/p44 MAPK responses.

In contrast to proinflammatory cytokines such as TNFα, growth factorreceptor tyrosine kinases are generally mitogenic in fibroblasts andstimulate the p42/p44 MAPK pathway. The bFGF receptor possessesintrinsic tyrosine kinase activity and is present on L929 cells. FIG. 26demonstrates that bFGF stimulates a robust activation of MAPK in L929cells. Concentrations of 0.25-0.5 ng/ml of bFGF gave maximal stimulationof MAPK activity. Fractionation of stimulated cell lysates by MonoQ fastpressure liquid chromatography indicated that both p42 and p44 MAPK wereactivated by bFGF (not shown). Activation of the MAPK pathway bytyrosine kinases involves Ras and the Raf serine-threonine proteinkinases. Immunoblotting demonstrated that B-Raf and C-Raf are expressedin L929 cells (not shown). Treatment of L929 cells with bFGF resulted inthe activation of both B-Raf and C-Raf as measured by their ability tophosphorylate a recombinant kinase-inactive MEK-1 protein (Gardner, A.M., Lange-Carter, C. A., Vaillancourt, R. R., and Johnson, G. L. (1994)Meth. Enzymol. 238:258-270). MEK-1 is the protein kinase phosphorylatedand activated by Raf, which in turn phosphorylates MAPK on both atyrosine and threonine resulting in MAPK activation (Crews, C. M.,Allesandrini, A., and Erikson, R. L. (1992) Science 258:478-480; Crews,C. M., and Erikson, R. L. (1992) Proc. Natl. Acad. Sci. (USA)89:8205-8209; Nakielny, S., et al. (1992) EMBO J. 11:2123-2129; Seger,R., et al. (1992) J. Biol. Chem. 267:14373-14381). In contrast, TNFαdoes not significantly activate either isoform of Raf in L929 cells.

bFGF and TNFα independently regulate cytoplasmic protein kinase cascadesFIG. 27 demonstrates that 1 ng/ml TNFα has only modest stimulatoryeffects on MAPK activity (panel B) and 2.5 ng/ml bFGF has little or noeffect on JNK activity (Panel A). These concentrations of bFGF and TNFαgive maximal activation of MAPK and JNK, respectively. Co-stimulation ofL929 cells with bFGF, at concentrations that show partial protectionagainst TNFα-mediated killing, did not alter the magnitude of JNKactivation in response to TNFα. Similarly, co-stimulation of L929 cellswith TNFα, at concentrations capable of causing cell death, had littleor no effect on bFGF stimulation of MAPK activity (Panel B). Thus, inrelation to JNK and MAPK, TNFα and bFGF receptors independently regulatethe activity of these two sequential protein kinase pathways in L929cells.Inducible expression of inhibitory and activated Ras influencesapoptosis Ras activation is required for many of the phenotypicresponses resulting from the activation of tyrosine kinases. Signalingby the bFGF receptor involves several different effector pathwaysincluding Ras activation. To test the involvement of Ras in the bFGFprotective response, the Lac Switch inducible expression system (seeMethods) was used to control the expression of inhibitory N17 Ras andconstitutively activated V12 Ras in L929 cells. FIG. 28 shows thefunctional consequence of expressing inhibitory N17 Ras or activated V12Ras on MAPK and JNK activation in response to bFGF and TNFα,respectively. IPTG-regulated expression of the HA epitope-tagged Rasmutants (N17 and V12 Ras) is shown in Panel D. Expression of N17 Rassignificantly blunted bFGF stimulation of MAPK (Panel A), but had noeffect on TNF stimulation of JNK (Panel C). With two independent clones,expression of V12 Ras did not constitutively activate the MAPK pathway,but did appear to enhance bFGF stimulation of MAPK (Panel B). V12 Rasexpression also had no effect on TNFα stimulation of JNK activity (PanelC). Similar results were found with independent L929 cell clonesindicating the responses were the result of specific mutant Rasexpression.

Expression of N17 Ras did not affect TNFα induced apoptosis of L929cells; N17 Ras did, however, markedly inhibit the ability of bFGF toprotect cells against TNFα-mediated cell death. These findings indicatedthat functional Ras signaling is not required for the TNF α-inducedapoptotic response, but is required for the protective action of bFGF.Strikingly, constitutively activated V12 Ras has markedly enhancedTNFα-stimulated apoptosis, but had little or no effect on the apoptoticindex of L929 cells in the absence of TNFα. This observation indicatesthat V12 Ras is functional in L929 cells, despite the fact MAPK is notconstitutively activated in this cell line and implies that activatedRas likely regulates pathways in addition to MAPK that are involved inapoptosis. Co-stimulation with bFGF and TNFα resulted in a diminishedapoptotic response relative to TNFα alone in V12 Ras expressing cells,indicating that bFGF pathways required for protection against TNFαstimulated cell death were functional in these cells (FIG. 29). Thus,inhibitory Ras expression prevented bFGF protective responses andactivated Ras enhanced TNFα killing. The results suggest multipleRas-dependent events are involved in controlling apoptosis and the roleof Ras signaling can be either positive or negative in regulating thephenotypic response to cytokines such as TNFα.

Inhibition of MEK and MAPK stimulation prevents bFGF protection fromapoptosis The Parke-Davis compound, PD #098059 inhibits the dualspecificity protein kinase, MEK-1, which specifically activates p42/p44MAPK (Alesssi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel,A. R. (1995) J. Biol. Chem. 270:27489-27494). PD #098059 did not inhibitJNK kinase or the activation of JNK (not shown). Pretreatment of L929cells with PD #098059 inhibited bFGF stimulation of MAPK activity (FIG.30A). The PD #098059 compound had no effect on TNFα-mediated apoptosisbut inhibited the protective action of bFGF (FIG. 30B). Thus, MEKactivation of MAPK is required for bFGF protection against TNFα-mediatedapoptosis. Interestingly, the phosphatidylinositol 3-kinase inhibitor,wortmannin, did not influence the cell death response to TNFα nor did itinhibit the protective response to bFGF (not shown). Treatment of L929cells with wortmannin had no effect on the ability of bFGF to stimulateMAPK activity. Apparently, phosphatidylinositol 3-kinase activity is notrequired for the action of either TNFα or bFGF on the control of thecell death program L929 cells.

TNFα induces apoptosis of L929 cells and bFGF is protective against thiscell death response. Our results indicate that the activation of JNK inresponse to TNFα stimulation of L929 cells is not sufficient for theinduction of cell death. TNFα maximally stimulates JNK activity in thepresence of bFGF concentrations that are capable of protecting againstcell death. Signals in addition to JNK activation must be involved inthe TNFα-mediated death response. The bFGF protective response was onlypartial in that not all the cells were prevented from dying in responseto TNFα treatment. This may, in part, be related to cell cycle dependentsignaling by TNFα and bFGF; the L929 cells used in these studies wereasynchronous so that we can not rule out this possibility. Our findingsalso demonstrate that Ras is involved in integrating responses thatcontrol apoptosis. Expression of activated or inhibitory Ras influencesTNFα killing of L929 cells. The mechanism for enhanced TNFα killing ofL929 cells resulting from V12 Ras expression is unclear, although it hasbeen observed in C3H mouse fibroblasts as well (Fernandez, A., et al.(1994) Oncogene 9:2009-2017). It may involve an alteration in theexpression of specific genes such as c-Jun, c-Fos and c-Myc which appearto be involved in both growth and apoptotic responses (Westwick, J., etal. (1994) J. Biol. Chem. 269:26396-6401; Pulverer, B. J., et al. (1991)Nature 353:670-674; Seth, A., et al. (1991) J. Biol. Chem.266:23521-23524; Evan, G. I., et al. (1992) Cell 69:119-128; Gupta, S.,Seth, A., and Davis, R. J. (1993) Proc. Natl. Acad. Sci. (USA)90:3216-3220; Klefstrom, J., et al. (1994) EMBO J. 13:5442-5450; Shi,Y., et al (1992) Science 257:212-214; Janicke, R. U., Lee, F. H. H., andPorter, A. G. (1994) Mol. Cell. Biol. 14:5661-5670; (Harrington, E. A.,et al. (1994) EMBO J. 13:3286-3295). In contrast, the effect ofinhibitory N17 Ras appears to primarily be the inhibition of MAPKactivation in response to bFGF. This finding is substantiated by theloss of bFGF protection against TNFα-mediated apoptosis by the MEKinhibitor PD #098059. Studies using the fungal metabolite, wortmannin,demonstrated that phosphatidylinositol 3-kinase was not involved in bFGFprotection against apoptosis in L929 cells.

Recently, it was demonstrated using PC12 cells that the JNK pathway wasinvolved in mediating apoptosis in response to serum deprivation andthat activation of the MAPK pathway was protective against serumdeprivation (Xia, Z., et al. (1995) Science 270:1326-1331).Phosphatidylinositol 3-kinase activity has also been reported to benecessary to protect PC12 cells from serum deprivation induced apoptosis(Yao, R., and Gooper, G. M. (1995) Science 267:2003-2006).Interestingly, the expression of N17 Ras protected PC12 cells from nervegrowth factor withdrawal induced apoptosis (Ferrari, G., and Greene, L.A. (1994) EMBO J. 13:5922-5928). The findings indicated that N17 Rasmaintained PC12 cells in a quiescent state that allowed them to survivein the absence of trophic factors. Removal of trophic factors from PC12cells appeared to induce an aberrant proliferative response thatresulted in apoptosis. Our findings using N17 Ras expression in L929cells contrast with those in PC12 cells. TNFα induced apoptosis ingrowing L929 cells, N17 Ras expression did not affect the apoptoticresponse, while V12 Ras expression significantly enhanced apoptosis.Thus, the involvement of Ras dependent signaling on apoptotic responsesof cycling versus quiescent cells may be quite different.

In human B cells, crosslinking of surface IgM stimulated a host ofsignaling pathways including MAPK but not JNK and resulted in apoptosis(Sakata, N., Patel, H., Aruffo, A., Johnson, G. L., and Gelfand, E. W.(1995) J. Biol. Chem. 270:30823-30828). CD40, a member of the TNFreceptor family, activated JNK while rescuing B cells from anti-IgMmediated apoptosis (Sakata, N., Patel, H., Aruffo, A., Johnson, G. L.,and Gelfand, E. W. (1995) J. Biol. Chem. 270:30823-30828). Thus, inhuman B cells MAPK activation is insufficient to protect againstapoptosis and signals including the stimulation of JNK are generatedduring a protective response. Clearly, the integration of multiplesignals appears to be required for apoptosis.

The overlap of signals involved in committing cells to growth orapoptosis is also evident in many transformed cell types. Tumorsfrequently have a high growth rate, but also a high apoptotic index(Evan, G. I., et al. (1992) Cell 69:119-128; Fanidi, A., Harrington, E.A., and Evan, G. I. (1992) Nature 359:554-556). The growth rate issimply greater than the apoptotic rate so that the net result is tumorexpansion. In addition, transformed cells frequently have selectedmutations and growth factor autocrine loops to inhibit apoptosis. Forexample, Ras function has been shown to be involved in bothtransformation and protection against apoptosis in Bcr-Abl transformedcells (Cortey, D., Kadlec, L., and Pendergast, A. M. (1995) Mol. Cell.Biol. 15:5531-5541; Goga, A., et al. (1995) Cell 82:981-988).

Cumulatively, the results in different cell types indicate that it isthe integration of multiple signals from cytokines and growth factorsthat determines the commitment to apoptosis. Similarly, integration ofmultiple signals and not a single dominant signaling pathway is likelyinvolved in the commitment to growth or differentiation. The requirementfor signal integration may allow for specific checkpoints so that cellsdo not die or grow inappropriately. In this regard, cell systems wherespecific cytokines or growth factors are added or removed are mostrelevant in defining the integration of signals controlling growthversus death.

The implication of our findings is that it should be possible to definesignal pathways and their integration that controls apoptosis inspecific cell types. As these findings are further defined it will bepossible to develop strategies to selectively induce a celltype-specific apoptotic response. Development of gene therapy, cytokineand drug treatments may be possible to selectively promote the death ofundesirable cell populations in animals.

Example 31 This Example Illustrated the Translocation of MEKK1 and MEKK2in Response to EGF and TNFα

Swiss 3T3 cells were serum starved overnight and then treated for 10minutes with either EGF or TNFα. Cells were fixed and stained with anantibody specifically recognizing either MEKK1 or MEKK2. SecondaryFITC-conjugated anti-rabbit IgG antibody was used for staining.

The results indicated that MEKK1 was localized primarily in thecytoplasm. A weak plasma membrane staining was also evident. MEKK2 wasprimarily cytoplasmic with little or no plasma membrane staining.

Stimulation with EGF induced a dramatic translocation of MEKK1 to theplasma membrane. treatment of the cells with EGF did not effect thecellular localization of MEKK2. Stimulation of the cells with TNFαinduced a translocation of MEKK2 to the plasma membrane. TNFα had noeffect on the cellular localization of MEKK1. Both EGF and TNFαstimulate the Jun kinase (JNK) pathway but regulate different MEKKS. EGFselectively regulates MEKK1 and TNFα selectively regulates MEKK2. Thesignificance of this finding is the demonstration that growth factorreceptor tyrosine kinases and cytokine receptors of the TNF familyselectively and differentially regulate specific MEKK enzymes.

The foregoing description of the invention has been presented forpurposes of illustration and description. Further, the description isnot intended to limit the invention to the form disclosed herein.Consequently, variations and modifications commensurate with the aboveteachings, and the skill or knowledge in the relevant art are within thescope of the present invention. The preferred embodiment describedherein above is further intended to explain the best mode known ofpracticing the invention and to enable others skilled in the art toutilize the invention in various embodiments and with variousmodifications required by their particular applications or uses of theinvention. It is intended that the appended claims be construed toinclude alternate embodiments to the extent permitted by the prior art.

1. An isolated or recombinant MEK Kinase polypeptide comprising a MEKKinase amino acid sequence depicted in one or more of SEQ ID Nos: 6, 8,10, 12, or
 14. 2. A nucleic acid encoding a mammalian MEKK protein 3.The MEKK nucleic acid of claim 2, which nucleic acid comprises a codingsequence represented in one of SEQ ID Nos:5, 7, 9, 11, or
 13. 4. Thenucleic acid of claim 2, which nucleic acid encodes a MEKK polypeptidedesignated by one of SEQ ID Nos: 6, 8, 10, 12, or
 14. 5. A nucleic acidhomolog which hybridizes under stringent conditions with one or more ofSEQ ID Nos: 5, 7, 9, 11, or
 13. 6. A nucleic acid comprising anucleotide sequence at least 50% percent homologous with one of SEQ IDNos:5, 7, 9, 11, or 13, or complementary thereto.
 7. An nucleic acidwhich encodes a MEKK polypeptide, wherein said polypeptide (i)phosphorylates a MAP kinase kinase protein and (ii) binds to a rassuperfamily protein.
 8. The nucleic acid of claim 6, wherein saidnucleic acid encodes a polypeptide which comprises a MKK consensusbinding site.
 9. The nucleic acid of claim 6, wherein said MAP kinasekinase is selected from the group consisting of p42, p44, ERK1, ERK2,JNK1, JNK2, or p38 SAPK.
 10. The nucleic acid of claim 6, wherein saidnucleic acid encodes a polypeptide which comprises a rac/cdc42 bindingsite.
 11. The nucleic acid of claim 6, wherein said nucleic acid encodesa polypeptide which does not contain an SH2 or SH3 domain.
 12. Thenucleic acid of claim 6, wherein said nucleic acid encodes a polypeptidewhich comprises a proline rich SH3 binding motif.
 13. The nucleic acidof claim 6, wherein said nucleic acid encodes a polypeptide whichcomprises a Pleckstrin Homology Domain.
 14. The nucleic acid of claim 6,wherein said nucleic acid encodes a polypeptide which is capable ofregulating apoptosis in a cell.
 15. A nucleic acid which encodes apolypeptide which is capable of competitively inhibiting the activity ofa MEKK designated in one or more of SEQ ID Nos: 6, 8, 10, 12, or
 14. 16.The nucleic acid of claim 14, wherein said nucleic acid encodes apolypeptide which is at least 50% homologous with one of SEQ ID Nos: 6,8, 10, 12, or
 14. 17. An nucleic acid homolog encoding a truncated MEKKpolypeptide.
 18. The nucleic acid of claim 17, wherein said polypeptidecomprises a region having at least 50% homolog with the protein kinasecatalytic domain represented by amino acids 361-620 of SEQ ID No:6,361-620 of SEQ ID No:8, 366-626 of SEQ ID No:10, 631-890 or SEQ IDNo:12, or 1338-1597 of SEQ ID No:14.
 19. The nucleic acid of claim 17,wherein said polypeptide comprises a region having at least 50% homologwith the serine/threonine rich regulatory domain represented by aminoacids 1-360 of SEQ ID No:6, 1-360 of SEQ ID No:8, 1-365 of SEQ ID No:10,1-630 or SEQ ID No:12, or 1-1337 of SEQ ID No:14.
 20. An isolatednucleic acid which encodes a protein kinase catalytic domain representedin one of SEQ ID Nos: 6, 8, 10, 12, or
 14. 21. The nucleic acid of claim20, wherein said protein kinase catalytic domain comprises amino acids361-620 of SEQ ID No:6, 361-620 of SEQ ID No:8, 366-626 of SEQ ID No:10,631-890 or SEQ ID No:12, or 1338-1597 of SEQ ID No:
 14. 22. An isolatednucleic acid which encodes an NH2 regulatory domain represented in oneof SEQ ID Nos: 6, 8, 10, 12, or
 14. 23. The nucleic acid of claim 22,wherein said NH₂ regulatory domain comprises amino acids 1-360 of SEQ IDNo:6, 1-360 of SEQ ID No:8, 1-365 of SEQ ID No:10, 1-630 or SEQ IDNo:12, or 1-1337 of SEQ ID No:14.
 24. The nucleic acid of claim 15,wherein said polypeptide is a fusion protein further comprising, inaddition to said MEKK polypeptide, a second polypeptide sequence havingan amino acid sequence unrelated to said MEKK nucleic acid sequence. 25.The nucleic acid of claim 24, wherein said fusion protein includes, as asecond polypeptide sequence, a polypeptide which functions as adetectable label for detecting the presence of said fusion protein or asa matrix-binding domain for immobilizing said fusion protein.
 26. Thenucleic acid of claim 15, which nucleic acid hybridizes under stringentconditions to a nucleic acid probe having a sequence represented by atleast 60 consecutive nucleotides of sense or antisense of one or more ofSEQ ID Nos:5, 7, 9, 11, or
 13. 27. The nucleic acid of claim 15, furthercomprising a transcriptional regulatory sequence operably linked to saidnucleotide sequence so as to render said nucleic acid suitable for useas an expression vector.
 28. An expression vector, capable ofreplicating in at least one of a prokaryotic cell and eukaryotic cell,comprising the nucleic acid of claim
 15. 29. A host cell transfectedwith the expression vector of claim 28 and expressing said recombinantpolypeptide.
 30. A method of producing a recombinant MEKK polypeptidecomprising culturing the cell of claim 29 in a cell culture medium toexpress said recombinant polypeptide and isolating said recombinantpolypeptide from said cell culture.
 31. A transgenic animal having cellswhich harbor a transgene encoding a MEKK polypeptide, which animals arevertebrates.
 32. A transgenic animal having cells in which a gene for aMEKK is disrupted, which animals are vertebrates.
 33. A recombinanttransfection system, comprising: (i) a gene construct including thenucleic acid of claim 6 and operably linked to a transcriptionalregulatory sequence for causing expression of said MEKK polypeptide ineukaryotic cells, and (ii) a gene delivery composition for deliveringsaid gene construct to a cell and causing the cell to be transfectedwith said gene construct.
 34. The recombinant transfection system ofclaim 33, wherein the gene delivery composition is selected from a groupconsisting of a recombinant viral particle, a liposome, and apoly-cationic nucleic acid binding agent.
 35. A nucleic acid compositioncomprising a substantially purified oligonucleotide, saidoligonucleotide including a region of nucleotide sequence whichhybridizes under stringent conditions to at least 25 consecutivenucleotides of sense or antisense sequence of a vertebrate MEKK gene.36. The nucleic acid composition of claim 35, which oligonucleotidehybridizes under stringent conditions to at least 50 consecutivenucleotides of sense or antisense sequence of a vertebrate MEKK gene.37. The nucleic acid composition of claim 36, wherein saidoligonucleotide further comprises a label group attached thereto andable to be detected.
 38. The nucleic acid composition of claim 37,wherein said oligonucleotide has at least one non-hydrolyzable bondbetween two adjacent nucleotide subunits.
 39. A test kit for detectingcells which contain a MEKK mRNA transcript, comprising the nucleic acidcomposition of claim 37 for measuring, in a sample of cells, a level ofnucleic acid encoding a MEKK protein.
 40. A method for modulating one ormore of growth, differentiation, or survival of a mammalian cell saidcell possessing or engineered to posses MEKK substrates, comprisingtreating the cell with an effective amount of an agent which activatesor inactivates MEKK polypeptide thereby altering, relative to the cellin the absence of the agent, at least one of (i) rate of growth, (ii)differentiation, or (iii) survival of the cell.